<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2025 Workiva-->
<!--r:d4b72416-14c6-44a8-8543-b716c9263197,g:d1f029a7-4abf-40ec-ba31-088b27a79438-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://heartflow.com/role/CondensedConsolidatedStatementsofRedeemableConvertiblePreferredStockandStockholdersEquityDeficit" xlink:type="simple" xlink:href="htfl-20250930.xsd#CondensedConsolidatedStatementsofRedeemableConvertiblePreferredStockandStockholdersEquityDeficit"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/CondensedConsolidatedStatementsofRedeemableConvertiblePreferredStockandStockholdersEquityDeficit" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_26958e8c-a688-41ed-a7ce-81fbd06976b3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_26958e8c-a688-41ed-a7ce-81fbd06976b3" xlink:to="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_788514a0-3401-467d-85f8-bd3e327aa95d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_788514a0-3401-467d-85f8-bd3e327aa95d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_788514a0-3401-467d-85f8-bd3e327aa95d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_788514a0-3401-467d-85f8-bd3e327aa95d" xlink:to="loc_us-gaap_EquityComponentDomain_788514a0-3401-467d-85f8-bd3e327aa95d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_788514a0-3401-467d-85f8-bd3e327aa95d" xlink:to="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_b7ad749e-3be5-4620-83a1-1929feabccee" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:to="loc_us-gaap_CommonStockMember_b7ad749e-3be5-4620-83a1-1929feabccee" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_1e44ecb4-2d23-4207-9443-1edc984c2959" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_1e44ecb4-2d23-4207-9443-1edc984c2959" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_a90b097a-6168-4711-a0d0-45bbdb84c2c0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_a90b097a-6168-4711-a0d0-45bbdb84c2c0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_6e28f9c9-d182-4e6e-9f89-6d9ded2899d3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_9887a76b-77bd-4e6f-aae9-c0c70afddd07" xlink:to="loc_us-gaap_RetainedEarningsMember_6e28f9c9-d182-4e6e-9f89-6d9ded2899d3" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_1363151e-c820-4acf-a9ac-2bfd9988a07f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:to="loc_us-gaap_StatementClassOfStockAxis_1363151e-c820-4acf-a9ac-2bfd9988a07f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_1363151e-c820-4acf-a9ac-2bfd9988a07f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_1363151e-c820-4acf-a9ac-2bfd9988a07f" xlink:to="loc_us-gaap_ClassOfStockDomain_1363151e-c820-4acf-a9ac-2bfd9988a07f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_04d216ce-846a-46e8-a95c-772c663cd4d3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_1363151e-c820-4acf-a9ac-2bfd9988a07f" xlink:to="loc_us-gaap_ClassOfStockDomain_04d216ce-846a-46e8-a95c-772c663cd4d3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertiblePreferredStockMember_973da8ef-0250-42aa-9322-b06686bca3d5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_04d216ce-846a-46e8-a95c-772c663cd4d3" xlink:to="loc_us-gaap_ConvertiblePreferredStockMember_973da8ef-0250-42aa-9322-b06686bca3d5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_d4881585-728b-4a36-9e63-d05ff7a96777" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:to="loc_us-gaap_DebtInstrumentAxis_d4881585-728b-4a36-9e63-d05ff7a96777" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_d4881585-728b-4a36-9e63-d05ff7a96777_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_d4881585-728b-4a36-9e63-d05ff7a96777" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_d4881585-728b-4a36-9e63-d05ff7a96777_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_a64acd06-aa7f-4dc2-ae5d-97dd0d39490a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_d4881585-728b-4a36-9e63-d05ff7a96777" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_a64acd06-aa7f-4dc2-ae5d-97dd0d39490a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesMember_e3901b20-a650-425f-9662-a61c08dc8141" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_a64acd06-aa7f-4dc2-ae5d-97dd0d39490a" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesMember_e3901b20-a650-425f-9662-a61c08dc8141" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_fb5649d2-ae2e-4a24-a4fb-358bbd077a88" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_336afd71-4cc7-403c-ab19-b33768202791" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_bcf4a1b1-553e-4faf-9b66-69f0144b8a3c" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_336afd71-4cc7-403c-ab19-b33768202791" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_82717e52-303a-4df1-94a1-da9cdde2acd3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_336afd71-4cc7-403c-ab19-b33768202791" xlink:to="loc_us-gaap_ConvertibleDebtMember_82717e52-303a-4df1-94a1-da9cdde2acd3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInTemporaryEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_26958e8c-a688-41ed-a7ce-81fbd06976b3" xlink:to="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquitySharesOutstanding_555066ac-11ff-4577-b313-5d98adf2f242" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:to="loc_us-gaap_TemporaryEquitySharesOutstanding_555066ac-11ff-4577-b313-5d98adf2f242" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityCarryingAmountAttributableToParent_23200187-5114-42c2-a60d-9a092660a29d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityCarryingAmountAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:to="loc_us-gaap_TemporaryEquityCarryingAmountAttributableToParent_23200187-5114-42c2-a60d-9a092660a29d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_TemporaryEquitySharesConversionOfConvertibleSecurities_581f5183-9016-4fab-a8dc-41fc35d5c076" xlink:href="htfl-20250930.xsd#htfl_TemporaryEquitySharesConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:to="loc_htfl_TemporaryEquitySharesConversionOfConvertibleSecurities_581f5183-9016-4fab-a8dc-41fc35d5c076" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_TemporaryEquityValueConversionOfConvertibleSecurities_fdc8cf0b-d272-4e4f-92e3-8f4ae0ef3dab" xlink:href="htfl-20250930.xsd#htfl_TemporaryEquityValueConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInTemporaryEquityRollForward_ba906f56-cbfa-4226-9e73-35117685830c" xlink:to="loc_htfl_TemporaryEquityValueConversionOfConvertibleSecurities_fdc8cf0b-d272-4e4f-92e3-8f4ae0ef3dab" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquitySharesOutstanding_b4787814-7ba6-418d-88ab-c6bbbdb27b81" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityCarryingAmountAttributableToParent_945584b7-e965-492b-8e5f-4277f8ae4f6f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityCarryingAmountAttributableToParent"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_26958e8c-a688-41ed-a7ce-81fbd06976b3" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_daa24658-d7b1-4552-aa99-55a7ddee7979" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_CommonStockSharesOutstanding_daa24658-d7b1-4552-aa99-55a7ddee7979" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_5e57ee5c-4d9b-473b-bcce-cc94bbabce01" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockholdersEquity_5e57ee5c-4d9b-473b-bcce-cc94bbabce01" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_19d4c088-74f6-47d6-960b-18555a657ca6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_19d4c088-74f6-47d6-960b-18555a657ca6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities_5259a730-a371-4c14-b916-db73c95464db" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities_5259a730-a371-4c14-b916-db73c95464db" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_1da61f34-2f4b-412d-9e7f-0987cc427b67" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_1da61f34-2f4b-412d-9e7f-0987cc427b67" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_120c7734-e696-4870-a017-1c95c1bb6c66" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_120c7734-e696-4870-a017-1c95c1bb6c66" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_0ba469fe-13bb-4c64-95b2-81f437ef3b64" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_0ba469fe-13bb-4c64-95b2-81f437ef3b64" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_5ff5f26b-af02-4dc2-8b64-a2ca10e953a8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised_5ff5f26b-af02-4dc2-8b64-a2ca10e953a8" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_05bbe4d7-2a35-4787-9ae1-d09d7368c421" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_05bbe4d7-2a35-4787-9ae1-d09d7368c421" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_0a465332-5a9e-4b93-9e92-48026ce11bd1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_0a465332-5a9e-4b93-9e92-48026ce11bd1" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_fa8aa1ff-9945-4334-91c4-be1b12f733cd" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_d1737493-799a-41fb-ab40-bafe9cfc33fc" xlink:to="loc_us-gaap_NetIncomeLoss_fa8aa1ff-9945-4334-91c4-be1b12f733cd" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockSharesOutstanding_5cfe2a85-7f09-4886-9edf-f27c965efb7d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_2a48a64a-eb74-498c-8d39-aebd8144f908" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/BusinessOverviewDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#BusinessOverviewDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/BusinessOverviewDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_88e3f34f-1315-44aa-9822-6fdf18510c40" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_88e3f34f-1315-44aa-9822-6fdf18510c40" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_c515921b-ea00-47b2-8119-28716331524a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_88e3f34f-1315-44aa-9822-6fdf18510c40" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_c515921b-ea00-47b2-8119-28716331524a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_c515921b-ea00-47b2-8119-28716331524a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_c515921b-ea00-47b2-8119-28716331524a" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_c515921b-ea00-47b2-8119-28716331524a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_9c536368-35ff-424c-9d41-05ce9370dce4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_c515921b-ea00-47b2-8119-28716331524a" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_9c536368-35ff-424c-9d41-05ce9370dce4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IPOMember_6f22e325-2f37-43f9-bf6f-6affbf7c3566" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IPOMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_9c536368-35ff-424c-9d41-05ce9370dce4" xlink:to="loc_us-gaap_IPOMember_6f22e325-2f37-43f9-bf6f-6affbf7c3566" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OverAllotmentOptionMember_744ee14d-ba6d-481a-98b2-29513abf7367" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OverAllotmentOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_9c536368-35ff-424c-9d41-05ce9370dce4" xlink:to="loc_us-gaap_OverAllotmentOptionMember_744ee14d-ba6d-481a-98b2-29513abf7367" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_ConsolidatingTransactionConversionRatio_307b22af-0da4-4a5a-a3c6-dcbe0fe91f8e" xlink:href="htfl-20250930.xsd#htfl_ConsolidatingTransactionConversionRatio"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_htfl_ConsolidatingTransactionConversionRatio_307b22af-0da4-4a5a-a3c6-dcbe0fe91f8e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquityNoteStockSplitConversionRatio1_49854899-f867-42e9-a915-d623d98c3758" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquityNoteStockSplitConversionRatio1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_StockholdersEquityNoteStockSplitConversionRatio1_49854899-f867-42e9-a915-d623d98c3758" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_d922b1d9-23fc-45fd-a315-4ec093fbbf59" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction_d922b1d9-23fc-45fd-a315-4ec093fbbf59" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockPricePerShare_a8f45b76-e7f6-4fef-bed8-2ed62a4a5065" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockPricePerShare"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_SaleOfStockPricePerShare_a8f45b76-e7f6-4fef-bed8-2ed62a4a5065" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceInitialPublicOffering_9d42a7e1-4eb3-4e20-8ffd-9eee76c284d0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromIssuanceInitialPublicOffering"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_ProceedsFromIssuanceInitialPublicOffering_9d42a7e1-4eb3-4e20-8ffd-9eee76c284d0" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsOfStockIssuanceCosts_832a368f-2010-460d-a48d-23f60ea9e5ff" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfStockIssuanceCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_PaymentsOfStockIssuanceCosts_832a368f-2010-460d-a48d-23f60ea9e5ff" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsAccumulatedDeficit_ac3ede48-c980-4bc5-b1d0-e2781e93276d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsAccumulatedDeficit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_RetainedEarningsAccumulatedDeficit_ac3ede48-c980-4bc5-b1d0-e2781e93276d" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsAtCarryingValue_037d211b-3ef8-4736-b4d8-3ff078a87305" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_60ab47c3-667c-4a45-87cb-846c58fb4beb" xlink:to="loc_us-gaap_CashAndCashEquivalentsAtCarryingValue_037d211b-3ef8-4736-b4d8-3ff078a87305" xlink:type="arc" order="7"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/SummaryofSignificantAccountingPoliciesNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#SummaryofSignificantAccountingPoliciesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/SummaryofSignificantAccountingPoliciesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostAxis_7a230ea5-accc-42ac-aad6-00af32db4e21" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:to="loc_us-gaap_CapitalizedContractCostAxis_7a230ea5-accc-42ac-aad6-00af32db4e21" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostDomain_7a230ea5-accc-42ac-aad6-00af32db4e21_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CapitalizedContractCostAxis_7a230ea5-accc-42ac-aad6-00af32db4e21" xlink:to="loc_us-gaap_CapitalizedContractCostDomain_7a230ea5-accc-42ac-aad6-00af32db4e21_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostDomain_ca9f4b59-e528-4027-9150-27ffa87c633a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CapitalizedContractCostAxis_7a230ea5-accc-42ac-aad6-00af32db4e21" xlink:to="loc_us-gaap_CapitalizedContractCostDomain_ca9f4b59-e528-4027-9150-27ffa87c633a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_ImplementationAndOnboardingCostsMember_7e97d6d4-9090-47ac-86ba-4c66cd4927d1" xlink:href="htfl-20250930.xsd#htfl_ImplementationAndOnboardingCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostDomain_ca9f4b59-e528-4027-9150-27ffa87c633a" xlink:to="loc_htfl_ImplementationAndOnboardingCostsMember_7e97d6d4-9090-47ac-86ba-4c66cd4927d1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_NewSiteCommissionCostsMember_31af758d-446b-420c-8739-77bb2b854625" xlink:href="htfl-20250930.xsd#htfl_NewSiteCommissionCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostDomain_ca9f4b59-e528-4027-9150-27ffa87c633a" xlink:to="loc_htfl_NewSiteCommissionCostsMember_31af758d-446b-420c-8739-77bb2b854625" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_b591c5a8-418e-4bdf-8897-1b503b557a4e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:to="loc_us-gaap_AwardTypeAxis_b591c5a8-418e-4bdf-8897-1b503b557a4e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_b591c5a8-418e-4bdf-8897-1b503b557a4e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_b591c5a8-418e-4bdf-8897-1b503b557a4e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_b591c5a8-418e-4bdf-8897-1b503b557a4e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_16c96ee8-7991-496e-8205-dc9f73d619b0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_b591c5a8-418e-4bdf-8897-1b503b557a4e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_16c96ee8-7991-496e-8205-dc9f73d619b0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_d7dd58cf-4602-4ac7-ab66-4f489c5fda27" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_16c96ee8-7991-496e-8205-dc9f73d619b0" xlink:to="loc_us-gaap_EmployeeStockOptionMember_d7dd58cf-4602-4ac7-ab66-4f489c5fda27" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:to="loc_us-gaap_DebtInstrumentAxis_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_a7d0e886-e4d6-407e-bef0-39a210c2f3ef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_55bb3969-279e-4f15-8c32-fbbe9a6c4ffa" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_a7d0e886-e4d6-407e-bef0-39a210c2f3ef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesMember_23ada1af-3df9-46a3-b13d-68ea947087f7" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_a7d0e886-e4d6-407e-bef0-39a210c2f3ef" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesMember_23ada1af-3df9-46a3-b13d-68ea947087f7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_ca770955-1ec9-4710-9257-be8532d883d5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_3c2e6bfe-79f9-4b43-b12c-f0f6fe45588b" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_ca770955-1ec9-4710-9257-be8532d883d5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_2c1c41c9-7760-4b2a-a288-d4d3579fcf69" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_ca770955-1ec9-4710-9257-be8532d883d5" xlink:to="loc_us-gaap_ConvertibleDebtMember_2c1c41c9-7760-4b2a-a288-d4d3579fcf69" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_052a0919-a375-49ad-bfbd-7104dc2a79be" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_CapitalizedContractCostTable_ced6679e-21fb-4ebc-869f-dd3e1cd62cfb" xlink:to="loc_us-gaap_StatementClassOfStockAxis_052a0919-a375-49ad-bfbd-7104dc2a79be" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_052a0919-a375-49ad-bfbd-7104dc2a79be_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_052a0919-a375-49ad-bfbd-7104dc2a79be" xlink:to="loc_us-gaap_ClassOfStockDomain_052a0919-a375-49ad-bfbd-7104dc2a79be_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_0d1fa456-7a8d-433b-9b52-b769fcd769b6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_052a0919-a375-49ad-bfbd-7104dc2a79be" xlink:to="loc_us-gaap_ClassOfStockDomain_0d1fa456-7a8d-433b-9b52-b769fcd769b6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RedeemableConvertiblePreferredStockMember_68fa338e-52b5-43ea-9ed5-ff8d12729917" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_0d1fa456-7a8d-433b-9b52-b769fcd769b6" xlink:to="loc_us-gaap_RedeemableConvertiblePreferredStockMember_68fa338e-52b5-43ea-9ed5-ff8d12729917" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NumberOfReportableSegments_d65c73c2-f376-4e3e-b65a-e21bc20de004" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfReportableSegments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_NumberOfReportableSegments_d65c73c2-f376-4e3e-b65a-e21bc20de004" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NumberOfOperatingSegments_4d53a15c-c62b-4c1f-8c20-df0124e63c73" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfOperatingSegments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_NumberOfOperatingSegments_4d53a15c-c62b-4c1f-8c20-df0124e63c73" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DeferredOfferingCostsNoncurrent_c6ff763a-5c5e-4c8c-b9ab-c474deb5d142" xlink:href="htfl-20250930.xsd#htfl_DeferredOfferingCostsNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_htfl_DeferredOfferingCostsNoncurrent_c6ff763a-5c5e-4c8c-b9ab-c474deb5d142" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseTermOfContract_0ae74f5f-dcba-4d1c-bb88-b7b525d04ac3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseTermOfContract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_LesseeOperatingLeaseTermOfContract_0ae74f5f-dcba-4d1c-bb88-b7b525d04ac3" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_2b38181d-177b-4091-a33e-a4bf4841f8d2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_2b38181d-177b-4091-a33e-a4bf4841f8d2" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_34d407d2-8de3-4ab0-985c-e17db9b441b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_34d407d2-8de3-4ab0-985c-e17db9b441b9" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilities_6eac5ddc-c1bf-4819-ade0-b711353fb47d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_DerivativeLiabilities_6eac5ddc-c1bf-4819-ade0-b711353fb47d" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature_cba92e8b-f7ac-4e6e-9c65-5d3804850f5b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature_cba92e8b-f7ac-4e6e-9c65-5d3804850f5b" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_RevenueRecognitionCustomerContractPeriod_47b6cc05-e52c-46fe-bc5b-5024f94d7a64" xlink:href="htfl-20250930.xsd#htfl_RevenueRecognitionCustomerContractPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_htfl_RevenueRecognitionCustomerContractPeriod_47b6cc05-e52c-46fe-bc5b-5024f94d7a64" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedContractCostAmortizationPeriod_4b9592a5-583b-42ac-8677-bfb07e15f82c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAmortizationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_CapitalizedContractCostAmortizationPeriod_4b9592a5-583b-42ac-8677-bfb07e15f82c" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_d7f31361-56c9-49af-a1ef-9d72d497765d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_d7f31361-56c9-49af-a1ef-9d72d497765d" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ForeignCurrencyTransactionGainLossAfterTax_61aed232-6764-4d3f-9c9b-f090d8614943" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCurrencyTransactionGainLossAfterTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_ForeignCurrencyTransactionGainLossAfterTax_61aed232-6764-4d3f-9c9b-f090d8614943" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_1cb64e2f-a69e-42bc-b31d-cda7b8998700" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalizedContractCostLineItems_3002034f-1045-4276-9f05-e305311db1e8" xlink:to="loc_us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent_1cb64e2f-a69e-42bc-b31d-cda7b8998700" xlink:type="arc" order="12"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/SummaryofSignificantAccountingPoliciesScheduleofSegmentReportingInformationDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#SummaryofSignificantAccountingPoliciesScheduleofSegmentReportingInformationDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/SummaryofSignificantAccountingPoliciesScheduleofSegmentReportingInformationDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentReportingInformationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_40fb5636-2116-4fe9-9486-074bff6985fb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_40fb5636-2116-4fe9-9486-074bff6985fb" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementBusinessSegmentsAxis_174a2de9-11e8-4fe6-9641-680699cc9f9b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementBusinessSegmentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable_40fb5636-2116-4fe9-9486-074bff6985fb" xlink:to="loc_us-gaap_StatementBusinessSegmentsAxis_174a2de9-11e8-4fe6-9641-680699cc9f9b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_174a2de9-11e8-4fe6-9641-680699cc9f9b_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_174a2de9-11e8-4fe6-9641-680699cc9f9b" xlink:to="loc_us-gaap_SegmentDomain_174a2de9-11e8-4fe6-9641-680699cc9f9b_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SegmentDomain_f9f1cc8d-9d36-4eb1-88a1-6c8dc160d839" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SegmentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementBusinessSegmentsAxis_174a2de9-11e8-4fe6-9641-680699cc9f9b" xlink:to="loc_us-gaap_SegmentDomain_f9f1cc8d-9d36-4eb1-88a1-6c8dc160d839" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_NonInvasiveCADDetectionSolutionsMember_21461d61-cfb2-4b1d-ad4b-bf2918e2f912" xlink:href="htfl-20250930.xsd#htfl_NonInvasiveCADDetectionSolutionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentDomain_f9f1cc8d-9d36-4eb1-88a1-6c8dc160d839" xlink:to="loc_htfl_NonInvasiveCADDetectionSolutionsMember_21461d61-cfb2-4b1d-ad4b-bf2918e2f912" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_82be86bd-b5a3-4520-b7d7-8dfbd5d07f10" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_82be86bd-b5a3-4520-b7d7-8dfbd5d07f10" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract_ba0c0679-d351-44a5-bede-e669354facca" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract_ba0c0679-d351-44a5-bede-e669354facca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfRevenue_4c0d092e-bc77-4fe1-9ca0-1d90e43725f0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfRevenue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract_ba0c0679-d351-44a5-bede-e669354facca" xlink:to="loc_us-gaap_CostOfRevenue_4c0d092e-bc77-4fe1-9ca0-1d90e43725f0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_500f1f6b-69b0-47ac-9b6c-4e27ac8361f7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract_ba0c0679-d351-44a5-bede-e669354facca" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_500f1f6b-69b0-47ac-9b6c-4e27ac8361f7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_ResearchAndDevelopmentExpenseOtherThanRegulatoryAndClinicalExpense_b0535d23-6289-40cd-8a8a-309b3b0ec26d" xlink:href="htfl-20250930.xsd#htfl_ResearchAndDevelopmentExpenseOtherThanRegulatoryAndClinicalExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_500f1f6b-69b0-47ac-9b6c-4e27ac8361f7" xlink:to="loc_htfl_ResearchAndDevelopmentExpenseOtherThanRegulatoryAndClinicalExpense_b0535d23-6289-40cd-8a8a-309b3b0ec26d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_RegulatoryAndClinicalExpense_0f8aa7f2-2210-47f8-9559-e2a8f0d32a1e" xlink:href="htfl-20250930.xsd#htfl_RegulatoryAndClinicalExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ResearchAndDevelopmentExpenseAbstract_500f1f6b-69b0-47ac-9b6c-4e27ac8361f7" xlink:to="loc_htfl_RegulatoryAndClinicalExpense_0f8aa7f2-2210-47f8-9559-e2a8f0d32a1e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingGeneralAndAdministrativeExpenseAbstract_c54970a0-61f5-40ae-a950-cb41177315a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingGeneralAndAdministrativeExpenseAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ReconciliationFromSegmentTotalsToConsolidatedAbstract_ba0c0679-d351-44a5-bede-e669354facca" xlink:to="loc_us-gaap_SellingGeneralAndAdministrativeExpenseAbstract_c54970a0-61f5-40ae-a950-cb41177315a5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SellingExpense_5a03f939-e5be-459b-b004-698efb50d3ac" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SellingExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SellingGeneralAndAdministrativeExpenseAbstract_c54970a0-61f5-40ae-a950-cb41177315a5" xlink:to="loc_us-gaap_SellingExpense_5a03f939-e5be-459b-b004-698efb50d3ac" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MarketingExpense_b74cb32e-02e9-4f1c-a207-6cb1fae8dc61" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MarketingExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SellingGeneralAndAdministrativeExpenseAbstract_c54970a0-61f5-40ae-a950-cb41177315a5" xlink:to="loc_us-gaap_MarketingExpense_b74cb32e-02e9-4f1c-a207-6cb1fae8dc61" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpense_e9d454b2-69c8-4e84-9f3d-23a47c7d3ce6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SellingGeneralAndAdministrativeExpenseAbstract_c54970a0-61f5-40ae-a950-cb41177315a5" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpense_e9d454b2-69c8-4e84-9f3d-23a47c7d3ce6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingIncomeLoss_06b7fb82-ea9c-43d3-b858-3db2bd507998" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_OperatingIncomeLoss_06b7fb82-ea9c-43d3-b858-3db2bd507998" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NonoperatingIncomeExpense_f13a3a16-9be8-4131-b681-2174a567b1ce" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonoperatingIncomeExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_NonoperatingIncomeExpense_f13a3a16-9be8-4131-b681-2174a567b1ce" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeTaxExpenseBenefit_91f6a38b-9ae8-4505-bdc6-661ec634fd21" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeTaxExpenseBenefit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_IncomeTaxExpenseBenefit_91f6a38b-9ae8-4505-bdc6-661ec634fd21" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_cd124f36-21bc-4450-abec-acb69c83ff80" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SegmentReportingInformationLineItems_ced30258-810c-47ff-8f81-fd348e3afd01" xlink:to="loc_us-gaap_NetIncomeLoss_cd124f36-21bc-4450-abec-acb69c83ff80" xlink:type="arc" order="5"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/RevenueandContractBalancesScheduleofTotalRevenueFromCustomersByGeographicRegionDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#RevenueandContractBalancesScheduleofTotalRevenueFromCustomersByGeographicRegionDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/RevenueandContractBalancesScheduleofTotalRevenueFromCustomersByGeographicRegionDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueLineItems_0cdd499f-71fb-4803-a269-4104f4f0ab28" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DisaggregationOfRevenueTable_6c0ba66e-8068-4bf9-8c55-38e988eaa322" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_0cdd499f-71fb-4803-a269-4104f4f0ab28" xlink:to="loc_us-gaap_DisaggregationOfRevenueTable_6c0ba66e-8068-4bf9-8c55-38e988eaa322" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_fdf96b02-7155-4839-b8e0-e2d369462f29" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DisaggregationOfRevenueTable_6c0ba66e-8068-4bf9-8c55-38e988eaa322" xlink:to="loc_srt_StatementGeographicalAxis_fdf96b02-7155-4839-b8e0-e2d369462f29" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_fdf96b02-7155-4839-b8e0-e2d369462f29_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_fdf96b02-7155-4839-b8e0-e2d369462f29" xlink:to="loc_srt_SegmentGeographicalDomain_fdf96b02-7155-4839-b8e0-e2d369462f29_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_fdf96b02-7155-4839-b8e0-e2d369462f29" xlink:to="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_US_11324203-4913-46f8-8fd3-5dae072b176b" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:to="loc_country_US_11324203-4913-46f8-8fd3-5dae072b176b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_GB_90ac8783-a7a7-4faa-b1c8-0af55128b906" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_GB"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:to="loc_country_GB_90ac8783-a7a7-4faa-b1c8-0af55128b906" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_country_JP_b51a0372-b878-4c07-90ec-457e246ba34e" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_JP"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:to="loc_country_JP_b51a0372-b878-4c07-90ec-457e246ba34e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_RestOfEuropeMember_ee17ab7a-8194-4977-86a9-0b48ba73f326" xlink:href="htfl-20250930.xsd#htfl_RestOfEuropeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1896b03f-5710-4042-aa3d-e5d04e825d2a" xlink:to="loc_htfl_RestOfEuropeMember_ee17ab7a-8194-4977-86a9-0b48ba73f326" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_dc9ecc86-50fa-400c-8bd6-4ef746bcc058" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DisaggregationOfRevenueLineItems_0cdd499f-71fb-4803-a269-4104f4f0ab28" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax_dc9ecc86-50fa-400c-8bd6-4ef746bcc058" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/FairValueMeasurementScheduleofFinancialAssetsandLiabilitiesMeasuredatFairValueonaRecurringBasisDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_73ffd7ea-90ac-4974-9599-eecbec5d5136" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_52e27e3a-910f-4bf9-8a1f-8b080dca441c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_73ffd7ea-90ac-4974-9599-eecbec5d5136" xlink:to="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_52e27e3a-910f-4bf9-8a1f-8b080dca441c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_434889fc-bca8-4f6a-87c4-12ded7bdc015" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable_52e27e3a-910f-4bf9-8a1f-8b080dca441c" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_434889fc-bca8-4f6a-87c4-12ded7bdc015" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_434889fc-bca8-4f6a-87c4-12ded7bdc015_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_434889fc-bca8-4f6a-87c4-12ded7bdc015" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_434889fc-bca8-4f6a-87c4-12ded7bdc015_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2ce56778-6a6a-40d3-aa79-a2c9760ccf11" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_434889fc-bca8-4f6a-87c4-12ded7bdc015" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2ce56778-6a6a-40d3-aa79-a2c9760ccf11" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_d11b8664-d777-494e-9c8a-87fdb92eae0b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2ce56778-6a6a-40d3-aa79-a2c9760ccf11" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_d11b8664-d777-494e-9c8a-87fdb92eae0b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_7fbb4028-4cda-481d-a81a-344eb0280933" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2ce56778-6a6a-40d3-aa79-a2c9760ccf11" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_7fbb4028-4cda-481d-a81a-344eb0280933" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel3Member_2bf00061-6a2a-4825-99ed-874f77e7325d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel3Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2ce56778-6a6a-40d3-aa79-a2c9760ccf11" xlink:to="loc_us-gaap_FairValueInputsLevel3Member_2bf00061-6a2a-4825-99ed-874f77e7325d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsFairValueDisclosureAbstract_9726a493-20cb-4e71-a425-9cc8dc8c614d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsFairValueDisclosureAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_73ffd7ea-90ac-4974-9599-eecbec5d5136" xlink:to="loc_us-gaap_AssetsFairValueDisclosureAbstract_9726a493-20cb-4e71-a425-9cc8dc8c614d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_7bcd0ff2-0267-4740-a2d6-dc3629923efe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashAndCashEquivalentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsFairValueDisclosureAbstract_9726a493-20cb-4e71-a425-9cc8dc8c614d" xlink:to="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_7bcd0ff2-0267-4740-a2d6-dc3629923efe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsFairValueDisclosure_9247f232-5d92-4a47-aeee-a9b3183043c7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AssetsFairValueDisclosureAbstract_9726a493-20cb-4e71-a425-9cc8dc8c614d" xlink:to="loc_us-gaap_AssetsFairValueDisclosure_9247f232-5d92-4a47-aeee-a9b3183043c7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesFairValueDisclosureAbstract_e0ff2006-999d-4c03-a506-e2ca1df57a63" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesFairValueDisclosureAbstract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems_73ffd7ea-90ac-4974-9599-eecbec5d5136" xlink:to="loc_us-gaap_LiabilitiesFairValueDisclosureAbstract_e0ff2006-999d-4c03-a506-e2ca1df57a63" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstanding_dc058070-6486-458b-8df2-540dfb9d4684" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantsAndRightsOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesFairValueDisclosureAbstract_e0ff2006-999d-4c03-a506-e2ca1df57a63" xlink:to="loc_us-gaap_WarrantsAndRightsOutstanding_dc058070-6486-458b-8df2-540dfb9d4684" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LiabilitiesFairValueDisclosure_3faed82f-985b-4cc0-87d8-2254a6d45113" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LiabilitiesFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LiabilitiesFairValueDisclosureAbstract_e0ff2006-999d-4c03-a506-e2ca1df57a63" xlink:to="loc_us-gaap_LiabilitiesFairValueDisclosure_3faed82f-985b-4cc0-87d8-2254a6d45113" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/FairValueMeasurementScheduleofSignificantUnobservableInputsLevel3andtheChangeinFairValueDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#FairValueMeasurementScheduleofSignificantUnobservableInputsLevel3andtheChangeinFairValueDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/FairValueMeasurementScheduleofSignificantUnobservableInputsLevel3andtheChangeinFairValueDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_857373d6-6c0f-41db-9f91-fa215a1dc108" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable_f2c9884a-7f27-49e3-a519-ca49b3d9ee6a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_857373d6-6c0f-41db-9f91-fa215a1dc108" xlink:to="loc_us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable_f2c9884a-7f27-49e3-a519-ca49b3d9ee6a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByLiabilityClassAxis_4d3ba964-2bee-457c-8a2b-7adca6424d0e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByLiabilityClassAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable_f2c9884a-7f27-49e3-a519-ca49b3d9ee6a" xlink:to="loc_us-gaap_FairValueByLiabilityClassAxis_4d3ba964-2bee-457c-8a2b-7adca6424d0e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_4d3ba964-2bee-457c-8a2b-7adca6424d0e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByLiabilityClassAxis_4d3ba964-2bee-457c-8a2b-7adca6424d0e" xlink:to="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_4d3ba964-2bee-457c-8a2b-7adca6424d0e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_6f094c63-29d7-4d7a-886c-9a497f2a0a11" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByLiabilityClassAxis_4d3ba964-2bee-457c-8a2b-7adca6424d0e" xlink:to="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_6f094c63-29d7-4d7a-886c-9a497f2a0a11" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockWarrantLiabilityMember_aaf801c7-d8c5-4151-949b-ed57c2bc358c" xlink:href="htfl-20250930.xsd#htfl_CommonStockWarrantLiabilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_6f094c63-29d7-4d7a-886c-9a497f2a0a11" xlink:to="loc_htfl_CommonStockWarrantLiabilityMember_aaf801c7-d8c5-4151-949b-ed57c2bc358c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_TermLoanDerivativeLiabilityMember_34a63e6b-835e-4c0c-b810-1cb1ca4f046e" xlink:href="htfl-20250930.xsd#htfl_TermLoanDerivativeLiabilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_6f094c63-29d7-4d7a-886c-9a497f2a0a11" xlink:to="loc_htfl_TermLoanDerivativeLiabilityMember_34a63e6b-835e-4c0c-b810-1cb1ca4f046e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertibleNotesDerivativeLiabilityMember_c00a10b4-6215-458d-ad88-5da9b054325c" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertibleNotesDerivativeLiabilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_6f094c63-29d7-4d7a-886c-9a497f2a0a11" xlink:to="loc_htfl_A2025ConvertibleNotesDerivativeLiabilityMember_c00a10b4-6215-458d-ad88-5da9b054325c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems_857373d6-6c0f-41db-9f91-fa215a1dc108" xlink:to="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue_21e58d91-0880-43bc-b665-4230c66acbd1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:to="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue_21e58d91-0880-43bc-b665-4230c66acbd1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues_7b9b78c3-9f7e-4018-81a5-246b7287a28f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:to="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues_7b9b78c3-9f7e-4018-81a5-246b7287a28f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings_41524301-87d8-4dce-acc2-33a77947d9c7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:to="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings_41524301-87d8-4dce-acc2-33a77947d9c7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityDerecognitionRecognition_378b313c-53ad-4374-ba13-0b5a6d985e98" xlink:href="htfl-20250930.xsd#htfl_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityDerecognitionRecognition"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward_c769cee6-56a2-411f-988f-18064a3a3a43" xlink:to="loc_htfl_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityDerecognitionRecognition_378b313c-53ad-4374-ba13-0b5a6d985e98" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue_3fefbca9-62ff-48bb-9f5f-900aa948a79c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/BalanceSheetComponentsScheduleofPropertyandEquipmentDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#BalanceSheetComponentsScheduleofPropertyandEquipmentDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/BalanceSheetComponentsScheduleofPropertyandEquipmentDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentLineItems_a4d2ffd4-daaa-4bb0-8f37-5d0f245a3c76" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_ce7c4f85-5d53-4ced-90f3-23ac2bb03433" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_a4d2ffd4-daaa-4bb0-8f37-5d0f245a3c76" xlink:to="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_ce7c4f85-5d53-4ced-90f3-23ac2bb03433" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_935e90ca-f9ee-411d-a737-467550fbc2f2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_ce7c4f85-5d53-4ced-90f3-23ac2bb03433" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_935e90ca-f9ee-411d-a737-467550fbc2f2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_935e90ca-f9ee-411d-a737-467550fbc2f2_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_935e90ca-f9ee-411d-a737-467550fbc2f2" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_935e90ca-f9ee-411d-a737-467550fbc2f2_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_935e90ca-f9ee-411d-a737-467550fbc2f2" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_ComputerEquipmentAndSoftwareMember_ac1467ec-af69-4fb2-b209-1bd55fde593a" xlink:href="htfl-20250930.xsd#htfl_ComputerEquipmentAndSoftwareMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:to="loc_htfl_ComputerEquipmentAndSoftwareMember_ac1467ec-af69-4fb2-b209-1bd55fde593a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FurnitureAndFixturesMember_6049cde4-669d-4fd3-a24d-55e65c101346" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:to="loc_us-gaap_FurnitureAndFixturesMember_6049cde4-669d-4fd3-a24d-55e65c101346" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_dea31860-fe46-41ed-99ac-2def013e0a77" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:to="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_dea31860-fe46-41ed-99ac-2def013e0a77" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LeaseholdImprovementsMember_802b3217-cd0a-4c13-80f4-56456c9592e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:to="loc_us-gaap_LeaseholdImprovementsMember_802b3217-cd0a-4c13-80f4-56456c9592e7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConstructionInProgressMember_70f13adc-b3c1-48b9-94de-c6179e06fe08" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConstructionInProgressMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_bc630ce5-a440-4aad-95b9-822cae29b8f4" xlink:to="loc_us-gaap_ConstructionInProgressMember_70f13adc-b3c1-48b9-94de-c6179e06fe08" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentGross_ffeefbfe-f405-41a7-b30a-bcdc9da7a520" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_a4d2ffd4-daaa-4bb0-8f37-5d0f245a3c76" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentGross_ffeefbfe-f405-41a7-b30a-bcdc9da7a520" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_1cc76a83-aa4c-4317-9e54-b4789b0e6e94" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_a4d2ffd4-daaa-4bb0-8f37-5d0f245a3c76" xlink:to="loc_us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment_1cc76a83-aa4c-4317-9e54-b4789b0e6e94" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentNet_fbc4a548-4ae2-4dad-8940-d626a324c591" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_a4d2ffd4-daaa-4bb0-8f37-5d0f245a3c76" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentNet_fbc4a548-4ae2-4dad-8940-d626a324c591" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/BalanceSheetComponentsNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#BalanceSheetComponentsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/BalanceSheetComponentsNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_a1d9a614-898f-41b1-909b-0a66eac49555" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:to="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_a1d9a614-898f-41b1-909b-0a66eac49555" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_81496d07-6764-47bc-b8c4-6a914c708d05" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfPropertyPlantAndEquipmentTable_a1d9a614-898f-41b1-909b-0a66eac49555" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_81496d07-6764-47bc-b8c4-6a914c708d05" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_81496d07-6764-47bc-b8c4-6a914c708d05_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_81496d07-6764-47bc-b8c4-6a914c708d05" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_81496d07-6764-47bc-b8c4-6a914c708d05_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_c68593e8-3daa-456e-9bd0-f46da177c1cb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentByTypeAxis_81496d07-6764-47bc-b8c4-6a914c708d05" xlink:to="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_c68593e8-3daa-456e-9bd0-f46da177c1cb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_aa2d2296-0683-4e40-846b-3b8634ded81c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_c68593e8-3daa-456e-9bd0-f46da177c1cb" xlink:to="loc_us-gaap_SoftwareAndSoftwareDevelopmentCostsMember_aa2d2296-0683-4e40-846b-3b8634ded81c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember_73c74916-2ecd-4e8b-8e16-c6f42e06737b" xlink:href="htfl-20250930.xsd#htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentTypeDomain_c68593e8-3daa-456e-9bd0-f46da177c1cb" xlink:to="loc_htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember_73c74916-2ecd-4e8b-8e16-c6f42e06737b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedComputerSoftwareAdditions_a11c7bf0-fa2e-4751-8718-2e285af03207" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedComputerSoftwareAdditions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:to="loc_us-gaap_CapitalizedComputerSoftwareAdditions_a11c7bf0-fa2e-4751-8718-2e285af03207" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_ed15ebcb-74b5-4580-8ff2-68eef7ad4f23" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_ed15ebcb-74b5-4580-8ff2-68eef7ad4f23" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalizedComputerSoftwareAmortization1_528136cf-9512-4e57-98a0-51f177edac70" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedComputerSoftwareAmortization1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:to="loc_us-gaap_CapitalizedComputerSoftwareAmortization1_528136cf-9512-4e57-98a0-51f177edac70" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DepreciationDepletionAndAmortization_d386e56d-d3a6-4433-93ea-9a9a33059b4e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DepreciationDepletionAndAmortization"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PropertyPlantAndEquipmentLineItems_b46b274d-9179-426f-8504-3720a1e71f87" xlink:to="loc_us-gaap_DepreciationDepletionAndAmortization_d386e56d-d3a6-4433-93ea-9a9a33059b4e" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/LeasesNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#LeasesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/LeasesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeLeaseDescriptionTable_16854498-24d4-485f-8b39-e920501f2fda" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_LesseeLeaseDescriptionTable_16854498-24d4-485f-8b39-e920501f2fda" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_412dbb97-e738-4f51-b2e2-4c8db7f1394a" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LesseeLeaseDescriptionTable_16854498-24d4-485f-8b39-e920501f2fda" xlink:to="loc_srt_StatementGeographicalAxis_412dbb97-e738-4f51-b2e2-4c8db7f1394a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_412dbb97-e738-4f51-b2e2-4c8db7f1394a_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_412dbb97-e738-4f51-b2e2-4c8db7f1394a" xlink:to="loc_srt_SegmentGeographicalDomain_412dbb97-e738-4f51-b2e2-4c8db7f1394a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_412dbb97-e738-4f51-b2e2-4c8db7f1394a" xlink:to="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_MountainViewCaliforniaMember_783b376b-0518-4dd0-9672-ffcf55d082b2" xlink:href="htfl-20250930.xsd#htfl_MountainViewCaliforniaMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:to="loc_htfl_MountainViewCaliforniaMember_783b376b-0518-4dd0-9672-ffcf55d082b2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SantaRosaCaliforniaMember_46f53546-b4a0-483a-8680-2cb757daf60e" xlink:href="htfl-20250930.xsd#htfl_SantaRosaCaliforniaMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:to="loc_htfl_SantaRosaCaliforniaMember_46f53546-b4a0-483a-8680-2cb757daf60e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_AustinTexasMember_4c7dc152-78cb-47ed-9f9a-022b7962f523" xlink:href="htfl-20250930.xsd#htfl_AustinTexasMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:to="loc_htfl_AustinTexasMember_4c7dc152-78cb-47ed-9f9a-022b7962f523" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_TokyoJapanMember_03d90e00-76ef-4919-9161-9ec8525d645e" xlink:href="htfl-20250930.xsd#htfl_TokyoJapanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_1d81f853-68aa-41d8-b964-844ab62eff7b" xlink:to="loc_htfl_TokyoJapanMember_03d90e00-76ef-4919-9161-9ec8525d645e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetRentableArea_69441882-eac5-4411-9f3d-666850a1268b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetRentableArea"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_NetRentableArea_69441882-eac5-4411-9f3d-666850a1268b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashNoncurrent_86051b4b-544d-40c1-8a3b-6440a7aae078" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCashNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_RestrictedCashNoncurrent_86051b4b-544d-40c1-8a3b-6440a7aae078" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseLeaseNotYetCommencedTermOfContract1_bf379fb2-77d7-4a3c-a57e-2ab035f3442f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseLeaseNotYetCommencedTermOfContract1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_LesseeOperatingLeaseLeaseNotYetCommencedTermOfContract1_bf379fb2-77d7-4a3c-a57e-2ab035f3442f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SecurityDeposit_6290505f-7bd7-420a-b030-6db17a6117e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SecurityDeposit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_SecurityDeposit_6290505f-7bd7-420a-b030-6db17a6117e7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseLiability_7aa3d603-eac5-4109-ac60-31da76b4620e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_OperatingLeaseLiability_7aa3d603-eac5-4109-ac60-31da76b4620e" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeaseRightOfUseAsset_b68840d4-b65f-4b80-9e0f-a0e5f022c3f4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseRightOfUseAsset"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_OperatingLeaseRightOfUseAsset_b68840d4-b65f-4b80-9e0f-a0e5f022c3f4" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseRenewalTerm_b6b86275-08b6-4073-8cf2-26bef2281668" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_LesseeOperatingLeaseRenewalTerm_b6b86275-08b6-4073-8cf2-26bef2281668" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_LesseeOperatingLeaseNumberOfRenewalOptions_04664af1-e482-446d-b34c-7c67a49b06d5" xlink:href="htfl-20250930.xsd#htfl_LesseeOperatingLeaseNumberOfRenewalOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_htfl_LesseeOperatingLeaseNumberOfRenewalOptions_04664af1-e482-446d-b34c-7c67a49b06d5" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_LesseeOperatingLeaseExtendedRenewalTerm_f0b43878-4e48-4675-9117-200019dfdd56" xlink:href="htfl-20250930.xsd#htfl_LesseeOperatingLeaseExtendedRenewalTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_htfl_LesseeOperatingLeaseExtendedRenewalTerm_f0b43878-4e48-4675-9117-200019dfdd56" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_LesseeOperatingLeaseNumberOfContracts_97021fd1-d0b9-4bd8-b02e-ea579f6b0795" xlink:href="htfl-20250930.xsd#htfl_LesseeOperatingLeaseNumberOfContracts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_htfl_LesseeOperatingLeaseNumberOfContracts_97021fd1-d0b9-4bd8-b02e-ea579f6b0795" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingLeasePayments_48733b22-f9ee-4a56-b9f2-fcfbf70ba804" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeasePayments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LesseeLeaseDescriptionLineItems_f4d3c60c-73db-4a0c-a3c4-f7ae52bdb9cf" xlink:to="loc_us-gaap_OperatingLeasePayments_48733b22-f9ee-4a56-b9f2-fcfbf70ba804" xlink:type="arc" order="10"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/CommitmentandContingenciesNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#CommitmentandContingenciesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/CommitmentandContingenciesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingenciesTable_796ebe99-ebaa-4f30-9b41-c2077faff93c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_LossContingenciesTable_796ebe99-ebaa-4f30-9b41-c2077faff93c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_2c61c958-b3a0-4a2b-a149-4036b1955312" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LossContingenciesTable_796ebe99-ebaa-4f30-9b41-c2077faff93c" xlink:to="loc_srt_RangeAxis_2c61c958-b3a0-4a2b-a149-4036b1955312" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_2c61c958-b3a0-4a2b-a149-4036b1955312_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_2c61c958-b3a0-4a2b-a149-4036b1955312" xlink:to="loc_srt_RangeMember_2c61c958-b3a0-4a2b-a149-4036b1955312_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_7ad1e18b-3be0-474e-a0bc-5c825d47900c" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_2c61c958-b3a0-4a2b-a149-4036b1955312" xlink:to="loc_srt_RangeMember_7ad1e18b-3be0-474e-a0bc-5c825d47900c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_4c056513-6b5b-43ac-8c18-c3b2df0a85ea" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_7ad1e18b-3be0-474e-a0bc-5c825d47900c" xlink:to="loc_srt_MinimumMember_4c056513-6b5b-43ac-8c18-c3b2df0a85ea" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractualObligation_9a2e6a35-0cb8-45f1-a51a-c418ac89921d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractualObligation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_ContractualObligation_9a2e6a35-0cb8-45f1-a51a-c418ac89921d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RoyaltyExpense_e897b008-c9d8-463c-a33c-9e8358e98fd8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RoyaltyExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_RoyaltyExpense_e897b008-c9d8-463c-a33c-9e8358e98fd8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermPurchaseCommitmentAmount_631ba448-6f85-4c42-bba0-d7f7aaf212fc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongTermPurchaseCommitmentAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_LongTermPurchaseCommitmentAmount_631ba448-6f85-4c42-bba0-d7f7aaf212fc" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermPurchaseCommitmentPeriod_ff723738-d794-4982-a936-f43e26fc8d8e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermPurchaseCommitmentPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_LongtermPurchaseCommitmentPeriod_ff723738-d794-4982-a936-f43e26fc8d8e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LossContingencyAccrualAtCarryingValue_53b8c94f-42e8-43d3-8e0f-dcbf474b1172" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingencyAccrualAtCarryingValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LossContingenciesLineItems_96494d68-73b7-4990-9fbe-0d53f8df98fe" xlink:to="loc_us-gaap_LossContingencyAccrualAtCarryingValue_53b8c94f-42e8-43d3-8e0f-dcbf474b1172" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/TermLoan2024CreditAgreementDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#TermLoan2024CreditAgreementDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/TermLoan2024CreditAgreementDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_57f8d513-0cf5-4567-b9c4-6cb5dfbe5f6a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_ce0c05a7-2542-4328-b3db-b1ea40eba030" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_57f8d513-0cf5-4567-b9c4-6cb5dfbe5f6a" xlink:to="loc_us-gaap_DebtInstrumentTable_ce0c05a7-2542-4328-b3db-b1ea40eba030" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_3c7a560c-f9d1-4365-a4fc-752f100b00fc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_ce0c05a7-2542-4328-b3db-b1ea40eba030" xlink:to="loc_us-gaap_DebtInstrumentAxis_3c7a560c-f9d1-4365-a4fc-752f100b00fc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_3c7a560c-f9d1-4365-a4fc-752f100b00fc_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_3c7a560c-f9d1-4365-a4fc-752f100b00fc" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_3c7a560c-f9d1-4365-a4fc-752f100b00fc_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_dd66e3c3-353e-450f-b71b-55dff60d6856" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_3c7a560c-f9d1-4365-a4fc-752f100b00fc" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_dd66e3c3-353e-450f-b71b-55dff60d6856" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_dd2e2502-c3c5-444d-b3bc-fc2415f2e43c" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_dd66e3c3-353e-450f-b71b-55dff60d6856" xlink:to="loc_htfl_A2024TermLoanMember_dd2e2502-c3c5-444d-b3bc-fc2415f2e43c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_743a56c6-2078-4279-a231-449af9e249c8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_ce0c05a7-2542-4328-b3db-b1ea40eba030" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_743a56c6-2078-4279-a231-449af9e249c8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_743a56c6-2078-4279-a231-449af9e249c8_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_743a56c6-2078-4279-a231-449af9e249c8" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_743a56c6-2078-4279-a231-449af9e249c8_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_af85b408-e4fa-465e-8bfa-329d689347f3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_743a56c6-2078-4279-a231-449af9e249c8" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_af85b408-e4fa-465e-8bfa-329d689347f3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_69727d30-444a-4a89-b4a9-4b45a41c76b4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_af85b408-e4fa-465e-8bfa-329d689347f3" xlink:to="loc_us-gaap_ConvertibleDebtMember_69727d30-444a-4a89-b4a9-4b45a41c76b4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityAxis_d2c9e6e1-fe76-4cef-b568-b3d39d15076d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_ce0c05a7-2542-4328-b3db-b1ea40eba030" xlink:to="loc_us-gaap_CreditFacilityAxis_d2c9e6e1-fe76-4cef-b568-b3d39d15076d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_d2c9e6e1-fe76-4cef-b568-b3d39d15076d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CreditFacilityAxis_d2c9e6e1-fe76-4cef-b568-b3d39d15076d" xlink:to="loc_us-gaap_CreditFacilityDomain_d2c9e6e1-fe76-4cef-b568-b3d39d15076d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_ef040ee1-42c5-4f9e-8097-3336e431e765" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CreditFacilityAxis_d2c9e6e1-fe76-4cef-b568-b3d39d15076d" xlink:to="loc_us-gaap_CreditFacilityDomain_ef040ee1-42c5-4f9e-8097-3336e431e765" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditMember_7a49dd23-1b7a-4e63-8fce-8d49ec886178" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_ef040ee1-42c5-4f9e-8097-3336e431e765" xlink:to="loc_us-gaap_LineOfCreditMember_7a49dd23-1b7a-4e63-8fce-8d49ec886178" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_2c7dcac0-663a-46ce-a215-8d7d1ab8ef7b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_57f8d513-0cf5-4567-b9c4-6cb5dfbe5f6a" xlink:to="loc_us-gaap_LineOfCreditFacilityMaximumBorrowingCapacity_2c7dcac0-663a-46ce-a215-8d7d1ab8ef7b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromConvertibleDebt_f1b65a07-4412-45da-8d35-c2b36c8ef10f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromConvertibleDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_57f8d513-0cf5-4567-b9c4-6cb5dfbe5f6a" xlink:to="loc_us-gaap_ProceedsFromConvertibleDebt_f1b65a07-4412-45da-8d35-c2b36c8ef10f" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/TermLoanPrepaymentTermsandOtherFeesDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#TermLoanPrepaymentTermsandOtherFeesDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/TermLoanPrepaymentTermsandOtherFeesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PrepaymentFeeAxis_2eda5b0c-bad7-4f7a-85fb-47b27b24045c" xlink:href="htfl-20250930.xsd#htfl_PrepaymentFeeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_htfl_PrepaymentFeeAxis_2eda5b0c-bad7-4f7a-85fb-47b27b24045c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PrepaymentFeeDomain_2eda5b0c-bad7-4f7a-85fb-47b27b24045c_default" xlink:href="htfl-20250930.xsd#htfl_PrepaymentFeeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_htfl_PrepaymentFeeAxis_2eda5b0c-bad7-4f7a-85fb-47b27b24045c" xlink:to="loc_htfl_PrepaymentFeeDomain_2eda5b0c-bad7-4f7a-85fb-47b27b24045c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PrepaymentFeeDomain_4a20a6b5-bff4-497e-b541-3a2e582d44bb" xlink:href="htfl-20250930.xsd#htfl_PrepaymentFeeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_htfl_PrepaymentFeeAxis_2eda5b0c-bad7-4f7a-85fb-47b27b24045c" xlink:to="loc_htfl_PrepaymentFeeDomain_4a20a6b5-bff4-497e-b541-3a2e582d44bb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PrepaymentsAfterSecondAnniversaryMember_68c02320-c06f-4e67-91ff-3a09fd80c899" xlink:href="htfl-20250930.xsd#htfl_PrepaymentsAfterSecondAnniversaryMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_htfl_PrepaymentFeeDomain_4a20a6b5-bff4-497e-b541-3a2e582d44bb" xlink:to="loc_htfl_PrepaymentsAfterSecondAnniversaryMember_68c02320-c06f-4e67-91ff-3a09fd80c899" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PrepaymentsOnAndAfterThirdAnniversaryMember_b4754b08-0144-4289-9ae0-577ed400e802" xlink:href="htfl-20250930.xsd#htfl_PrepaymentsOnAndAfterThirdAnniversaryMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_htfl_PrepaymentFeeDomain_4a20a6b5-bff4-497e-b541-3a2e582d44bb" xlink:to="loc_htfl_PrepaymentsOnAndAfterThirdAnniversaryMember_b4754b08-0144-4289-9ae0-577ed400e802" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_821121ef-3d3a-4d17-bcc6-2ef242976abf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_us-gaap_DebtInstrumentAxis_821121ef-3d3a-4d17-bcc6-2ef242976abf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_821121ef-3d3a-4d17-bcc6-2ef242976abf_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_821121ef-3d3a-4d17-bcc6-2ef242976abf" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_821121ef-3d3a-4d17-bcc6-2ef242976abf_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_e37584a7-162a-4e9d-9145-743e38b60c6b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_821121ef-3d3a-4d17-bcc6-2ef242976abf" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_e37584a7-162a-4e9d-9145-743e38b60c6b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_5af53c7c-7234-400f-9f82-9279ef0ca951" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_e37584a7-162a-4e9d-9145-743e38b60c6b" xlink:to="loc_htfl_A2024TermLoanMember_5af53c7c-7234-400f-9f82-9279ef0ca951" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024CreditAgreementMember_f319acc5-6f91-40b9-bcbc-f22256ef2fa4" xlink:href="htfl-20250930.xsd#htfl_A2024CreditAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_e37584a7-162a-4e9d-9145-743e38b60c6b" xlink:to="loc_htfl_A2024CreditAgreementMember_f319acc5-6f91-40b9-bcbc-f22256ef2fa4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_94cd0646-2387-405d-92dc-5914b3d8514c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_94cd0646-2387-405d-92dc-5914b3d8514c" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_94cd0646-2387-405d-92dc-5914b3d8514c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_94cd0646-2387-405d-92dc-5914b3d8514c" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_94cd0646-2387-405d-92dc-5914b3d8514c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_15772e5e-40a0-4db9-a86b-190d32b722b8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_94cd0646-2387-405d-92dc-5914b3d8514c" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_15772e5e-40a0-4db9-a86b-190d32b722b8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditMember_56885e2d-df6f-4025-83ee-13f9bec80f0e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_15772e5e-40a0-4db9-a86b-190d32b722b8" xlink:to="loc_us-gaap_LineOfCreditMember_56885e2d-df6f-4025-83ee-13f9bec80f0e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_95e5cdde-30f9-4f49-8c29-cd80602ac403" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_95e5cdde-30f9-4f49-8c29-cd80602ac403" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_95e5cdde-30f9-4f49-8c29-cd80602ac403_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_95e5cdde-30f9-4f49-8c29-cd80602ac403" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_95e5cdde-30f9-4f49-8c29-cd80602ac403_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_f7be9c80-b07a-408c-ac5d-0a13c1994ca3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_95e5cdde-30f9-4f49-8c29-cd80602ac403" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_f7be9c80-b07a-408c-ac5d-0a13c1994ca3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IPOMember_9c16bc45-bfe3-4883-8224-c480920654dc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IPOMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_f7be9c80-b07a-408c-ac5d-0a13c1994ca3" xlink:to="loc_us-gaap_IPOMember_9c16bc45-bfe3-4883-8224-c480920654dc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityAxis_5a6464e2-1155-450a-8440-bddd3e8d3254" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_us-gaap_CreditFacilityAxis_5a6464e2-1155-450a-8440-bddd3e8d3254" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_5a6464e2-1155-450a-8440-bddd3e8d3254_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CreditFacilityAxis_5a6464e2-1155-450a-8440-bddd3e8d3254" xlink:to="loc_us-gaap_CreditFacilityDomain_5a6464e2-1155-450a-8440-bddd3e8d3254_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CreditFacilityDomain_5578e840-8ab7-434c-beeb-d831979c9522" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CreditFacilityDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CreditFacilityAxis_5a6464e2-1155-450a-8440-bddd3e8d3254" xlink:to="loc_us-gaap_CreditFacilityDomain_5578e840-8ab7-434c-beeb-d831979c9522" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_0d9d2180-4feb-43f8-98ce-224f50ae04e0" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CreditFacilityDomain_5578e840-8ab7-434c-beeb-d831979c9522" xlink:to="loc_htfl_A2024TermLoanMember_0d9d2180-4feb-43f8-98ce-224f50ae04e0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NatureOfExpenseAxis_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NatureOfExpenseAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_bbdaeb6d-3c0b-4b17-8c19-38e3591bd668" xlink:to="loc_us-gaap_NatureOfExpenseAxis_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_NatureOfExpenseAxis_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1" xlink:to="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_19f7864d-b451-46ee-800a-203cec5e7450" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_InterimPeriodCostsNotAllocableDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_NatureOfExpenseAxis_b1277f7d-cbb5-4da1-8f7c-bc21e5ce2bb1" xlink:to="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_19f7864d-b451-46ee-800a-203cec5e7450" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_UpfrontFeeMember_4a467956-2e00-4969-b843-2a683b7682b9" xlink:href="htfl-20250930.xsd#htfl_UpfrontFeeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_19f7864d-b451-46ee-800a-203cec5e7450" xlink:to="loc_htfl_UpfrontFeeMember_4a467956-2e00-4969-b843-2a683b7682b9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_AgentFeeMember_eb542a45-a006-4618-9e84-649eaeb0a4de" xlink:href="htfl-20250930.xsd#htfl_AgentFeeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_InterimPeriodCostsNotAllocableDomain_19f7864d-b451-46ee-800a-203cec5e7450" xlink:to="loc_htfl_AgentFeeMember_eb542a45-a006-4618-9e84-649eaeb0a4de" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentPrepaymentFeePercentage_dd6989f1-b7b5-4da0-b3c9-ff33b0b73283" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentPrepaymentFeePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentPrepaymentFeePercentage_dd6989f1-b7b5-4da0-b3c9-ff33b0b73283" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMaximum_fd282656-7a66-45ed-8777-fe67330405c0" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMaximum_fd282656-7a66-45ed-8777-fe67330405c0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMinimum_9438e0fa-563d-48df-ade9-514e938f1a61" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesMinimum_9438e0fa-563d-48df-ade9-514e938f1a61" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesIfUnderwritersExerciseOptionsMinimum_0d3b75c5-c5b9-4e53-8781-75f9b990656b" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesIfUnderwritersExerciseOptionsMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentCovenantRepaymentUponConsummationOfInitialPublicOfferingOrSpecialPurposeAcquisitionCompaniesIfUnderwritersExerciseOptionsMinimum_0d3b75c5-c5b9-4e53-8781-75f9b990656b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredFinanceCostsNet_96fb4fd6-f745-4c35-b825-5e62b051c86c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredFinanceCostsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_us-gaap_DeferredFinanceCostsNet_96fb4fd6-f745-4c35-b825-5e62b051c86c" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentExitFeePercentage_b0ef063a-dcf4-4507-886f-ef915c572952" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentExitFeePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentExitFeePercentage_b0ef063a-dcf4-4507-886f-ef915c572952" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentEarlyRepaymentFeePercentage_9f9e9417-10cc-40dd-a1d7-fdd3da44be22" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentEarlyRepaymentFeePercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentEarlyRepaymentFeePercentage_9f9e9417-10cc-40dd-a1d7-fdd3da44be22" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentEarlyRepaymentFeeAmount_8b8e3488-3057-4359-a0d8-74312d85aade" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentEarlyRepaymentFeeAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentEarlyRepaymentFeeAmount_8b8e3488-3057-4359-a0d8-74312d85aade" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentNumberOfQuarterlyInstallments_d92155b7-79e2-4dfc-b469-ca0ba3b8c25b" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentNumberOfQuarterlyInstallments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentNumberOfQuarterlyInstallments_d92155b7-79e2-4dfc-b469-ca0ba3b8c25b" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentEarlyRepaymentFeeQuarterlyInstallments_f3a13cdf-ac39-4f75-8be5-d814b0fdde71" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentEarlyRepaymentFeeQuarterlyInstallments"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentEarlyRepaymentFeeQuarterlyInstallments_f3a13cdf-ac39-4f75-8be5-d814b0fdde71" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentAgentFeeGross_ca9620a1-7e6c-45c4-8b83-12a957ef158c" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentAgentFeeGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentAgentFeeGross_ca9620a1-7e6c-45c4-8b83-12a957ef158c" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentPeriodicPaymentAgentFees_fffd05f6-2bb3-41a6-9b4c-f366957a280d" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentPeriodicPaymentAgentFees"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentPeriodicPaymentAgentFees_fffd05f6-2bb3-41a6-9b4c-f366957a280d" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentUpfrontFee_e1e74df1-7071-4bad-a904-e15e3b7d144c" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentUpfrontFee"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentUpfrontFee_e1e74df1-7071-4bad-a904-e15e3b7d144c" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PaymentsOfDebtExtinguishmentCosts_e6b4750e-f51a-4b4e-8882-a7707b27c6e4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfDebtExtinguishmentCosts"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_us-gaap_PaymentsOfDebtExtinguishmentCosts_e6b4750e-f51a-4b4e-8882-a7707b27c6e4" xlink:type="arc" order="13"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RepaymentsOfLongTermDebt_bfd3d2b4-1fee-453e-95fe-0fc8e50609fb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RepaymentsOfLongTermDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_us-gaap_RepaymentsOfLongTermDebt_bfd3d2b4-1fee-453e-95fe-0fc8e50609fb" xlink:type="arc" order="14"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentExitFeeAndEarlyPrepaymentFeeAmount_3ff70da5-69db-4642-893f-84553fb06181" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentExitFeeAndEarlyPrepaymentFeeAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_DebtInstrumentExitFeeAndEarlyPrepaymentFeeAmount_3ff70da5-69db-4642-893f-84553fb06181" xlink:type="arc" order="15"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentRepaidPrincipal_3a139c47-e75f-4d50-a7e8-191c588f7014" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentRepaidPrincipal"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_us-gaap_DebtInstrumentRepaidPrincipal_3a139c47-e75f-4d50-a7e8-191c588f7014" xlink:type="arc" order="16"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_RepaymentOfAccruedInterest_f21f714e-4a2c-473e-be3b-c1bdfa44b176" xlink:href="htfl-20250930.xsd#htfl_RepaymentOfAccruedInterest"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_9bec03d8-a859-469e-8273-87de03d54f97" xlink:to="loc_htfl_RepaymentOfAccruedInterest_f21f714e-4a2c-473e-be3b-c1bdfa44b176" xlink:type="arc" order="17"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/TermLoanInterestDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#TermLoanInterestDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/TermLoanInterestDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_8799ebbd-03b6-4dad-9259-299af47bd888" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_8799ebbd-03b6-4dad-9259-299af47bd888" xlink:to="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_7eaf7a76-2f10-4d85-ae62-0db8f372e5df" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:to="loc_us-gaap_DebtInstrumentAxis_7eaf7a76-2f10-4d85-ae62-0db8f372e5df" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_7eaf7a76-2f10-4d85-ae62-0db8f372e5df_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_7eaf7a76-2f10-4d85-ae62-0db8f372e5df" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_7eaf7a76-2f10-4d85-ae62-0db8f372e5df_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_07a9314a-a2d9-4ca4-be9f-5b5b9e75ed59" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_7eaf7a76-2f10-4d85-ae62-0db8f372e5df" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_07a9314a-a2d9-4ca4-be9f-5b5b9e75ed59" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_33bfbc6c-b4cf-49c8-b5ae-c71a77985d68" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_07a9314a-a2d9-4ca4-be9f-5b5b9e75ed59" xlink:to="loc_htfl_A2024TermLoanMember_33bfbc6c-b4cf-49c8-b5ae-c71a77985d68" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VariableRateAxis_d199fee4-7ef3-49c7-8eb8-5c055487e53e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:to="loc_us-gaap_VariableRateAxis_d199fee4-7ef3-49c7-8eb8-5c055487e53e" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VariableRateDomain_d199fee4-7ef3-49c7-8eb8-5c055487e53e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_VariableRateAxis_d199fee4-7ef3-49c7-8eb8-5c055487e53e" xlink:to="loc_us-gaap_VariableRateDomain_d199fee4-7ef3-49c7-8eb8-5c055487e53e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_VariableRateAxis_d199fee4-7ef3-49c7-8eb8-5c055487e53e" xlink:to="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BaseRateMember_d4e682a1-c584-4feb-8e24-073e8f6a8017" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BaseRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:to="loc_us-gaap_BaseRateMember_d4e682a1-c584-4feb-8e24-073e8f6a8017" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SecuredOvernightFinancingRateSofrMember_b8abf4a3-2817-4f2d-9d02-3205f7673b9c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SecuredOvernightFinancingRateSofrMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:to="loc_us-gaap_SecuredOvernightFinancingRateSofrMember_b8abf4a3-2817-4f2d-9d02-3205f7673b9c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PrimeRateMember_b0da9b76-1286-4f1a-b0ae-39edb7bf8f96" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrimeRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:to="loc_us-gaap_PrimeRateMember_b0da9b76-1286-4f1a-b0ae-39edb7bf8f96" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_FederalReserveBankOfNewYorkRateMember_01674b05-250e-4a0c-8691-7edd9103f59f" xlink:href="htfl-20250930.xsd#htfl_FederalReserveBankOfNewYorkRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:to="loc_htfl_FederalReserveBankOfNewYorkRateMember_01674b05-250e-4a0c-8691-7edd9103f59f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_OneMonthSecuredOvernightFinancingRateSOFRMember_9d50175f-666f-4d51-b2f4-40f5ac615fbf" xlink:href="htfl-20250930.xsd#htfl_OneMonthSecuredOvernightFinancingRateSOFRMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VariableRateDomain_0ac54e9d-fb53-49ee-bbbf-1bf1f96209b1" xlink:to="loc_htfl_OneMonthSecuredOvernightFinancingRateSOFRMember_9d50175f-666f-4d51-b2f4-40f5ac615fbf" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_027269d0-7bfd-423f-91e0-27ed3141e85f" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:to="loc_srt_RangeAxis_027269d0-7bfd-423f-91e0-27ed3141e85f" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_027269d0-7bfd-423f-91e0-27ed3141e85f_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_027269d0-7bfd-423f-91e0-27ed3141e85f" xlink:to="loc_srt_RangeMember_027269d0-7bfd-423f-91e0-27ed3141e85f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_db625cc3-6347-45ef-b0b3-ea75c3794175" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_027269d0-7bfd-423f-91e0-27ed3141e85f" xlink:to="loc_srt_RangeMember_db625cc3-6347-45ef-b0b3-ea75c3794175" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_6c43959b-0eb8-41d8-889c-3bb742531d1f" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_db625cc3-6347-45ef-b0b3-ea75c3794175" xlink:to="loc_srt_MaximumMember_6c43959b-0eb8-41d8-889c-3bb742531d1f" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_6d0b2f92-a20c-4102-8098-b0d38cbbdc2f" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_db625cc3-6347-45ef-b0b3-ea75c3794175" xlink:to="loc_srt_MinimumMember_6d0b2f92-a20c-4102-8098-b0d38cbbdc2f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_efdcaf4a-26ea-4f02-82d7-0aa904cfd234" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_101bd645-f34a-46a9-a631-4af371890502" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_efdcaf4a-26ea-4f02-82d7-0aa904cfd234" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_efdcaf4a-26ea-4f02-82d7-0aa904cfd234_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_efdcaf4a-26ea-4f02-82d7-0aa904cfd234" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_efdcaf4a-26ea-4f02-82d7-0aa904cfd234_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_2a520a59-1c38-493e-96d5-5319dcc0f8e2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_efdcaf4a-26ea-4f02-82d7-0aa904cfd234" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_2a520a59-1c38-493e-96d5-5319dcc0f8e2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditMember_18453449-8128-4f9c-9192-30447fdad13b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_2a520a59-1c38-493e-96d5-5319dcc0f8e2" xlink:to="loc_us-gaap_LineOfCreditMember_18453449-8128-4f9c-9192-30447fdad13b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentBasisSpreadOnVariableRate1_76539b3a-78b0-427f-ac73-d2e34d55632d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentBasisSpreadOnVariableRate1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_8799ebbd-03b6-4dad-9259-299af47bd888" xlink:to="loc_us-gaap_DebtInstrumentBasisSpreadOnVariableRate1_76539b3a-78b0-427f-ac73-d2e34d55632d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentInterestRatePaidInKind_8e5c0a8a-b1fb-4508-8ce2-41ac0cc158bc" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentInterestRatePaidInKind"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_8799ebbd-03b6-4dad-9259-299af47bd888" xlink:to="loc_htfl_DebtInstrumentInterestRatePaidInKind_8e5c0a8a-b1fb-4508-8ce2-41ac0cc158bc" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/TermLoanDebtIssuanceCostsandDebtDiscountDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#TermLoanDebtIssuanceCostsandDebtDiscountDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/TermLoanDebtIssuanceCostsandDebtDiscountDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_a7d14e8a-326b-4f1b-bda2-dc8bf50784b7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_459d6200-71bd-4b54-ac62-0e8affa97e59" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_a7d14e8a-326b-4f1b-bda2-dc8bf50784b7" xlink:to="loc_us-gaap_DebtInstrumentTable_459d6200-71bd-4b54-ac62-0e8affa97e59" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_74aaff2e-05c4-486e-a8dd-7c99061de00a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_459d6200-71bd-4b54-ac62-0e8affa97e59" xlink:to="loc_us-gaap_DebtInstrumentAxis_74aaff2e-05c4-486e-a8dd-7c99061de00a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_74aaff2e-05c4-486e-a8dd-7c99061de00a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_74aaff2e-05c4-486e-a8dd-7c99061de00a" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_74aaff2e-05c4-486e-a8dd-7c99061de00a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_558807aa-afcc-4a09-9266-cf69b5a09b8a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_74aaff2e-05c4-486e-a8dd-7c99061de00a" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_558807aa-afcc-4a09-9266-cf69b5a09b8a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_4a4bdbc4-0361-4566-b2ed-0da531d74b08" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_558807aa-afcc-4a09-9266-cf69b5a09b8a" xlink:to="loc_htfl_A2024TermLoanMember_4a4bdbc4-0361-4566-b2ed-0da531d74b08" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_459d6200-71bd-4b54-ac62-0e8affa97e59" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_510aacee-5358-4d46-8db9-acad00910d66" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_1bfcfa30-3dd1-4ef1-842f-2e170b2a29d8" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_510aacee-5358-4d46-8db9-acad00910d66" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditMember_ee517223-4289-4583-8729-4badfce40884" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_510aacee-5358-4d46-8db9-acad00910d66" xlink:to="loc_us-gaap_LineOfCreditMember_ee517223-4289-4583-8729-4badfce40884" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_e6074e44-6e3b-424c-8453-2f1c38d4f64a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_510aacee-5358-4d46-8db9-acad00910d66" xlink:to="loc_us-gaap_ConvertibleDebtMember_e6074e44-6e3b-424c-8453-2f1c38d4f64a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilities_f004ae84-a3d0-44b6-a8ec-e4ef7f61a261" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_a7d14e8a-326b-4f1b-bda2-dc8bf50784b7" xlink:to="loc_us-gaap_DerivativeLiabilities_f004ae84-a3d0-44b6-a8ec-e4ef7f61a261" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromConvertibleDebt_6ce375f2-6cf8-4789-b799-81a0dbec6dfc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromConvertibleDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_a7d14e8a-326b-4f1b-bda2-dc8bf50784b7" xlink:to="loc_us-gaap_ProceedsFromConvertibleDebt_6ce375f2-6cf8-4789-b799-81a0dbec6dfc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentProRataDebtDiscount_360b515d-49cc-4e78-b1dc-9d0d6c7dde1d" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentProRataDebtDiscount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_a7d14e8a-326b-4f1b-bda2-dc8bf50784b7" xlink:to="loc_htfl_DebtInstrumentProRataDebtDiscount_360b515d-49cc-4e78-b1dc-9d0d6c7dde1d" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/TermLoanScheduleofComponentsofTermLoanDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#TermLoanScheduleofComponentsofTermLoanDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/TermLoanScheduleofComponentsofTermLoanDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTable_9840cc23-c1ab-457a-941a-21775922aa13" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_us-gaap_DebtInstrumentTable_9840cc23-c1ab-457a-941a-21775922aa13" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_b0a1b7ef-1bc6-44e5-8886-c112d1af555a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_9840cc23-c1ab-457a-941a-21775922aa13" xlink:to="loc_us-gaap_DebtInstrumentAxis_b0a1b7ef-1bc6-44e5-8886-c112d1af555a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_b0a1b7ef-1bc6-44e5-8886-c112d1af555a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_b0a1b7ef-1bc6-44e5-8886-c112d1af555a" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_b0a1b7ef-1bc6-44e5-8886-c112d1af555a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_c8df887b-e722-4785-96cb-635e6a9010ca" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_b0a1b7ef-1bc6-44e5-8886-c112d1af555a" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_c8df887b-e722-4785-96cb-635e6a9010ca" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_b347699d-c33d-478a-ba8c-32a192ae4bac" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_c8df887b-e722-4785-96cb-635e6a9010ca" xlink:to="loc_htfl_A2024TermLoanMember_b347699d-c33d-478a-ba8c-32a192ae4bac" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_5a1b636f-9f3f-495c-a307-e4e402af1a18" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_DebtInstrumentTable_9840cc23-c1ab-457a-941a-21775922aa13" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_5a1b636f-9f3f-495c-a307-e4e402af1a18" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_5a1b636f-9f3f-495c-a307-e4e402af1a18_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_5a1b636f-9f3f-495c-a307-e4e402af1a18" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_5a1b636f-9f3f-495c-a307-e4e402af1a18_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_da11c439-87db-40b3-98c6-31e8a2aae747" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_5a1b636f-9f3f-495c-a307-e4e402af1a18" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_da11c439-87db-40b3-98c6-31e8a2aae747" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditMember_c0d1373b-f9db-4fbd-84d0-2c9be7d374d1" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_da11c439-87db-40b3-98c6-31e8a2aae747" xlink:to="loc_us-gaap_LineOfCreditMember_c0d1373b-f9db-4fbd-84d0-2c9be7d374d1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentCarryingAmount_5aec18a9-71ca-40d7-8bdd-26f1a7b31559" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentCarryingAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_us-gaap_DebtInstrumentCarryingAmount_5aec18a9-71ca-40d7-8bdd-26f1a7b31559" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentAccretedExitFee_908ed570-2e29-4b8f-8bdf-65a0d239ba41" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentAccretedExitFee"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_htfl_DebtInstrumentAccretedExitFee_908ed570-2e29-4b8f-8bdf-65a0d239ba41" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentUnamortizedDiscount_5128f2b2-a50d-46db-8148-73ed76a28e17" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentUnamortizedDiscount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_us-gaap_DebtInstrumentUnamortizedDiscount_5128f2b2-a50d-46db-8148-73ed76a28e17" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredFinanceCostsNet_370e0812-abb8-493c-a43b-0cd995d79960" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredFinanceCostsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_us-gaap_DeferredFinanceCostsNet_370e0812-abb8-493c-a43b-0cd995d79960" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongTermDebt_eabf3405-577a-4d99-b35f-1858866796be" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongTermDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentLineItems_474b581e-f3ad-4c6a-9e3e-a416099680b5" xlink:to="loc_us-gaap_LongTermDebt_eabf3405-577a-4d99-b35f-1858866796be" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/ConvertibleNotesNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#ConvertibleNotesNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/ConvertibleNotesNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LineOfCreditFacilityTable_8ce23143-3d57-47e8-8a48-f1a102f5b849" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_LineOfCreditFacilityTable_8ce23143-3d57-47e8-8a48-f1a102f5b849" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_0764cdb1-4de9-4fd8-a747-aa701227e698" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_8ce23143-3d57-47e8-8a48-f1a102f5b849" xlink:to="loc_us-gaap_DebtInstrumentAxis_0764cdb1-4de9-4fd8-a747-aa701227e698" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_0764cdb1-4de9-4fd8-a747-aa701227e698_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_0764cdb1-4de9-4fd8-a747-aa701227e698" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_0764cdb1-4de9-4fd8-a747-aa701227e698_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_3791dfe0-ff12-40af-bac8-c87107cea853" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_0764cdb1-4de9-4fd8-a747-aa701227e698" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_3791dfe0-ff12-40af-bac8-c87107cea853" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesMember_bc083978-7f99-4ec2-ab7c-aea948e78f29" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_3791dfe0-ff12-40af-bac8-c87107cea853" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesMember_bc083978-7f99-4ec2-ab7c-aea948e78f29" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesSettledInCashMember_6a687e39-c06a-448d-881b-20c2922ac675" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesSettledInCashMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_htfl_A2025ConvertiblePromissoryNotesMember_bc083978-7f99-4ec2-ab7c-aea948e78f29" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesSettledInCashMember_6a687e39-c06a-448d-881b-20c2922ac675" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember_46225d7e-7e28-4d80-95a5-f8436c68de43" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_htfl_A2025ConvertiblePromissoryNotesMember_bc083978-7f99-4ec2-ab7c-aea948e78f29" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember_46225d7e-7e28-4d80-95a5-f8436c68de43" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember_b83eb2cd-6871-46df-8cc0-f8a4ce701611" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_htfl_A2025ConvertiblePromissoryNotesMember_bc083978-7f99-4ec2-ab7c-aea948e78f29" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember_b83eb2cd-6871-46df-8cc0-f8a4ce701611" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_LineOfCreditFacilityTable_8ce23143-3d57-47e8-8a48-f1a102f5b849" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_59ea2daf-53ee-4f77-b301-7645234eb0c2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_2a3e4dd6-8fce-4796-8c17-50ce8a28f79d" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_59ea2daf-53ee-4f77-b301-7645234eb0c2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_552645f9-885c-4d30-9bca-7cd0e15306bb" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_59ea2daf-53ee-4f77-b301-7645234eb0c2" xlink:to="loc_us-gaap_ConvertibleDebtMember_552645f9-885c-4d30-9bca-7cd0e15306bb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_8bc2a8da-f38c-447c-ad4e-c0f4ed75a75b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_8bc2a8da-f38c-447c-ad4e-c0f4ed75a75b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation_e385e8f1-48c0-4284-ac52-e11ce56771ec" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation_e385e8f1-48c0-4284-ac52-e11ce56771ec" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtConversionConvertedInstrumentAmount1_c9e394a5-422b-46a3-9e9a-62669ace11a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtConversionConvertedInstrumentAmount1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_DebtConversionConvertedInstrumentAmount1_c9e394a5-422b-46a3-9e9a-62669ace11a5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromConvertibleDebt_736757d3-8c75-4987-aa72-2cf1ee37f75b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromConvertibleDebt"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_ProceedsFromConvertibleDebt_736757d3-8c75-4987-aa72-2cf1ee37f75b" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DeferredFinanceCostsNet_991d15c9-8102-4962-8ec0-f10b14b09ea5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredFinanceCostsNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_DeferredFinanceCostsNet_991d15c9-8102-4962-8ec0-f10b14b09ea5" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentTerm_4713883f-d263-4bfa-8002-263a7ee48036" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_DebtInstrumentTerm_4713883f-d263-4bfa-8002-263a7ee48036" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_4f587849-fc39-4dd4-8fc1-82069b44ea9e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_4f587849-fc39-4dd4-8fc1-82069b44ea9e" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharePrice_e99ed793-3587-4ad9-bb6a-2b47d8ef3753" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_SharePrice_e99ed793-3587-4ad9-bb6a-2b47d8ef3753" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentConversionPriceDiscountPercentage_cc23fffb-f3c6-42b7-a8b0-0608eb6f16d4" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentConversionPriceDiscountPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_htfl_DebtInstrumentConversionPriceDiscountPercentage_cc23fffb-f3c6-42b7-a8b0-0608eb6f16d4" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilities_0c8d0f0d-87f3-4e07-be16-b5157b3e8c9a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_DerivativeLiabilities_0c8d0f0d-87f3-4e07-be16-b5157b3e8c9a" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature_2dd8a724-f1c2-4607-bc3a-73a29d7274ca" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LineOfCreditFacilityLineItems_d4dbc1df-d1c1-447a-a135-675502d3c7d3" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature_2dd8a724-f1c2-4607-bc3a-73a29d7274ca" xlink:type="arc" order="10"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/RedeemableConvertiblePreferredStockScheduleofTemporaryEquityDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#RedeemableConvertiblePreferredStockScheduleofTemporaryEquityDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/RedeemableConvertiblePreferredStockScheduleofTemporaryEquityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityByClassOfStockTable_217ec1e2-883f-4aa2-b57a-5530650e2fd4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityByClassOfStockTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquityByClassOfStockTable_217ec1e2-883f-4aa2-b57a-5530650e2fd4" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_TemporaryEquityByClassOfStockTable_217ec1e2-883f-4aa2-b57a-5530650e2fd4" xlink:to="loc_us-gaap_StatementClassOfStockAxis_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8" xlink:to="loc_us-gaap_ClassOfStockDomain_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_71f28a2b-9f94-4eb8-9354-1ca46c83b8c8" xlink:to="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesARedeemableConvertiblePreferredStockMember_f3db05a8-4f48-48e2-9165-46af0994c636" xlink:href="htfl-20250930.xsd#htfl_SeriesARedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesARedeemableConvertiblePreferredStockMember_f3db05a8-4f48-48e2-9165-46af0994c636" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesB1RedeemableConvertiblePreferredStockMember_75b84b82-6972-4a3f-b63a-00be5baea0a9" xlink:href="htfl-20250930.xsd#htfl_SeriesB1RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesB1RedeemableConvertiblePreferredStockMember_75b84b82-6972-4a3f-b63a-00be5baea0a9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesB2RedeemableConvertiblePreferredStockMember_be8f6640-1725-4fe1-892d-bfd55f73d062" xlink:href="htfl-20250930.xsd#htfl_SeriesB2RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesB2RedeemableConvertiblePreferredStockMember_be8f6640-1725-4fe1-892d-bfd55f73d062" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesCRedeemableConvertiblePreferredStockMember_2e53a26c-a949-4129-89aa-e91e03711920" xlink:href="htfl-20250930.xsd#htfl_SeriesCRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesCRedeemableConvertiblePreferredStockMember_2e53a26c-a949-4129-89aa-e91e03711920" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesDRedeemableConvertiblePreferredStockMember_f239c28c-8571-45a3-8b65-a7fc692af3ff" xlink:href="htfl-20250930.xsd#htfl_SeriesDRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesDRedeemableConvertiblePreferredStockMember_f239c28c-8571-45a3-8b65-a7fc692af3ff" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesERedeemableConvertiblePreferredStockMember_79ee69c7-005e-4c16-93f6-2301cb8eef84" xlink:href="htfl-20250930.xsd#htfl_SeriesERedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesERedeemableConvertiblePreferredStockMember_79ee69c7-005e-4c16-93f6-2301cb8eef84" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesFRedeemableConvertiblePreferredStockMember_69f00654-e77f-47f5-a28f-67f49db8f6e7" xlink:href="htfl-20250930.xsd#htfl_SeriesFRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesFRedeemableConvertiblePreferredStockMember_69f00654-e77f-47f5-a28f-67f49db8f6e7" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesF1RedeemableConvertiblePreferredStockMember_9a9ff399-1bb7-46cb-83f7-994aff01ff88" xlink:href="htfl-20250930.xsd#htfl_SeriesF1RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_fcdc7861-8fdd-4f59-95a2-d2c51c74e708" xlink:to="loc_htfl_SeriesF1RedeemableConvertiblePreferredStockMember_9a9ff399-1bb7-46cb-83f7-994aff01ff88" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquitySharesAuthorized_87bf5740-496a-49f3-af42-de6b91df13a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesAuthorized"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquitySharesAuthorized_87bf5740-496a-49f3-af42-de6b91df13a5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquitySharesIssued_6fb79e0c-8d6a-484f-86c6-5939821621f2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesIssued"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquitySharesIssued_6fb79e0c-8d6a-484f-86c6-5939821621f2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquitySharesOutstanding_5451a8ea-50e0-4347-a825-f5a966e1e288" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquitySharesOutstanding_5451a8ea-50e0-4347-a825-f5a966e1e288" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityCarryingAmountAttributableToParent_eb4c84b3-52f1-48b2-b3f9-3b86b088284e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityCarryingAmountAttributableToParent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquityCarryingAmountAttributableToParent_eb4c84b3-52f1-48b2-b3f9-3b86b088284e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityLiquidationPreference_9029bbfa-3e84-492f-8948-e705b7f02ff7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityLiquidationPreference"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_aaad50e9-6281-4ba0-9f20-de488025362d" xlink:to="loc_us-gaap_TemporaryEquityLiquidationPreference_9029bbfa-3e84-492f-8948-e705b7f02ff7" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/RedeemableConvertiblePreferredStockNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#RedeemableConvertiblePreferredStockNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/RedeemableConvertiblePreferredStockNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityLineItems_8cb306e5-b967-435b-a5ce-bc86bdee059d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TemporaryEquityByClassOfStockTable_465eab47-6659-4c9c-8db0-f9bb51acfed2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityByClassOfStockTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_TemporaryEquityLineItems_8cb306e5-b967-435b-a5ce-bc86bdee059d" xlink:to="loc_us-gaap_TemporaryEquityByClassOfStockTable_465eab47-6659-4c9c-8db0-f9bb51acfed2" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_a778d5ba-e48e-4011-80a2-9932dab38c9a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_TemporaryEquityByClassOfStockTable_465eab47-6659-4c9c-8db0-f9bb51acfed2" xlink:to="loc_us-gaap_StatementClassOfStockAxis_a778d5ba-e48e-4011-80a2-9932dab38c9a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_a778d5ba-e48e-4011-80a2-9932dab38c9a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_a778d5ba-e48e-4011-80a2-9932dab38c9a" xlink:to="loc_us-gaap_ClassOfStockDomain_a778d5ba-e48e-4011-80a2-9932dab38c9a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_a778d5ba-e48e-4011-80a2-9932dab38c9a" xlink:to="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RedeemableConvertiblePreferredStockMember_160e9d98-076b-43cb-b18f-f56f76944a15" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_us-gaap_RedeemableConvertiblePreferredStockMember_160e9d98-076b-43cb-b18f-f56f76944a15" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesARedeemableConvertiblePreferredStockMember_e97e46c1-7c03-4a6f-9c31-6e45c2a89a94" xlink:href="htfl-20250930.xsd#htfl_SeriesARedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesARedeemableConvertiblePreferredStockMember_e97e46c1-7c03-4a6f-9c31-6e45c2a89a94" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesB1RedeemableConvertiblePreferredStockMember_f78c0cc2-9f17-453e-b954-490c8609cad8" xlink:href="htfl-20250930.xsd#htfl_SeriesB1RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesB1RedeemableConvertiblePreferredStockMember_f78c0cc2-9f17-453e-b954-490c8609cad8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesB2RedeemableConvertiblePreferredStockMember_c83695c3-ae4d-4e41-a531-f5c5666c53e5" xlink:href="htfl-20250930.xsd#htfl_SeriesB2RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesB2RedeemableConvertiblePreferredStockMember_c83695c3-ae4d-4e41-a531-f5c5666c53e5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesCRedeemableConvertiblePreferredStockMember_eeab1bcd-4d1f-46d1-b590-c6fe2f43b675" xlink:href="htfl-20250930.xsd#htfl_SeriesCRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesCRedeemableConvertiblePreferredStockMember_eeab1bcd-4d1f-46d1-b590-c6fe2f43b675" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesDRedeemableConvertiblePreferredStockMember_cceae914-c7a3-4019-8fd3-705390d86ded" xlink:href="htfl-20250930.xsd#htfl_SeriesDRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesDRedeemableConvertiblePreferredStockMember_cceae914-c7a3-4019-8fd3-705390d86ded" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesERedeemableConvertiblePreferredStockMember_62fde19e-b3a4-411c-957c-60c2faf54898" xlink:href="htfl-20250930.xsd#htfl_SeriesERedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesERedeemableConvertiblePreferredStockMember_62fde19e-b3a4-411c-957c-60c2faf54898" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesFRedeemableConvertiblePreferredStockMember_d6a2c20f-559a-4657-be14-a2eaf7fd77f1" xlink:href="htfl-20250930.xsd#htfl_SeriesFRedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesFRedeemableConvertiblePreferredStockMember_d6a2c20f-559a-4657-be14-a2eaf7fd77f1" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SeriesF1RedeemableConvertiblePreferredStockMember_1eda705c-161b-4b4c-8552-ad9e59970357" xlink:href="htfl-20250930.xsd#htfl_SeriesF1RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_3d75705a-f27d-477b-b233-030e1a78a73e" xlink:to="loc_htfl_SeriesF1RedeemableConvertiblePreferredStockMember_1eda705c-161b-4b4c-8552-ad9e59970357" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_5941070c-eea1-414d-aaed-d08ee6c5c95c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_8cb306e5-b967-435b-a5ce-bc86bdee059d" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities_5941070c-eea1-414d-aaed-d08ee6c5c95c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PreferredStockConvertibleConversionRatio_52b9bc1b-015d-4837-9693-88bc6ca0eb75" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PreferredStockConvertibleConversionRatio"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TemporaryEquityLineItems_8cb306e5-b967-435b-a5ce-bc86bdee059d" xlink:to="loc_us-gaap_PreferredStockConvertibleConversionRatio_52b9bc1b-015d-4837-9693-88bc6ca0eb75" xlink:type="arc" order="1"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/StockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#StockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/StockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockLineItems_8ecf69f1-336c-45a0-9491-c8d973f642ab" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfStockByClassTable_49808047-fbe1-43d6-a324-031d466dd469" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfStockByClassTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfStockLineItems_8ecf69f1-336c-45a0-9491-c8d973f642ab" xlink:to="loc_us-gaap_ScheduleOfStockByClassTable_49808047-fbe1-43d6-a324-031d466dd469" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementClassOfStockAxis_c7f37831-1917-4907-a1f6-126f75818c99" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_49808047-fbe1-43d6-a324-031d466dd469" xlink:to="loc_us-gaap_StatementClassOfStockAxis_c7f37831-1917-4907-a1f6-126f75818c99" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_c7f37831-1917-4907-a1f6-126f75818c99_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementClassOfStockAxis_c7f37831-1917-4907-a1f6-126f75818c99" xlink:to="loc_us-gaap_ClassOfStockDomain_c7f37831-1917-4907-a1f6-126f75818c99_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfStockDomain_b27f6123-4e89-4734-a81f-8fbad814006b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementClassOfStockAxis_c7f37831-1917-4907-a1f6-126f75818c99" xlink:to="loc_us-gaap_ClassOfStockDomain_b27f6123-4e89-4734-a81f-8fbad814006b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RedeemableConvertiblePreferredStockMember_16bb6947-9f5d-43c6-bfd0-19ccb7e002a5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_b27f6123-4e89-4734-a81f-8fbad814006b" xlink:to="loc_us-gaap_RedeemableConvertiblePreferredStockMember_16bb6947-9f5d-43c6-bfd0-19ccb7e002a5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_fada7931-1519-40a7-a7a8-384266bd8e0f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockDomain_b27f6123-4e89-4734-a81f-8fbad814006b" xlink:to="loc_us-gaap_WarrantMember_fada7931-1519-40a7-a7a8-384266bd8e0f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_d9c9e607-06a1-4123-bd8f-0357873ba8a7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfStockByClassTable_49808047-fbe1-43d6-a324-031d466dd469" xlink:to="loc_us-gaap_AwardTypeAxis_d9c9e607-06a1-4123-bd8f-0357873ba8a7" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_d9c9e607-06a1-4123-bd8f-0357873ba8a7_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_d9c9e607-06a1-4123-bd8f-0357873ba8a7" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_d9c9e607-06a1-4123-bd8f-0357873ba8a7_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0156db54-5cc9-4334-acf3-be0794da7b8a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_d9c9e607-06a1-4123-bd8f-0357873ba8a7" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0156db54-5cc9-4334-acf3-be0794da7b8a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_40ce357e-fdb6-4481-874f-71ab90006576" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0156db54-5cc9-4334-acf3-be0794da7b8a" xlink:to="loc_us-gaap_EmployeeStockOptionMember_40ce357e-fdb6-4481-874f-71ab90006576" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_029665c3-bbad-48b4-9458-c679705ad129" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0156db54-5cc9-4334-acf3-be0794da7b8a" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_029665c3-bbad-48b4-9458-c679705ad129" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SharesReservedForIssuanceMember_1c3d54c3-57c1-4ccc-9523-213616b475cb" xlink:href="htfl-20250930.xsd#htfl_SharesReservedForIssuanceMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_0156db54-5cc9-4334-acf3-be0794da7b8a" xlink:to="loc_htfl_SharesReservedForIssuanceMember_1c3d54c3-57c1-4ccc-9523-213616b475cb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_2431813a-b628-43e4-82b3-264faba0e1e7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfStockLineItems_8ecf69f1-336c-45a0-9491-c8d973f642ab" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_2431813a-b628-43e4-82b3-264faba0e1e7" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/CommonStockWarrantLiabilityNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#CommonStockWarrantLiabilityNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/CommonStockWarrantLiabilityNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_45dd94c8-037c-4a72-ace0-caf261e9bded" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_45dd94c8-037c-4a72-ace0-caf261e9bded" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_45dd94c8-037c-4a72-ace0-caf261e9bded" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_d814feed-7218-401e-93d8-4cd287f6d630" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_85d0c8ff-83a0-46a1-99ac-fc4acc7c599f" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_d814feed-7218-401e-93d8-4cd287f6d630" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockWarrantInitialTermLoanMember_a5ed15f4-ca0c-48f4-9bf5-4a60dbd6a7c2" xlink:href="htfl-20250930.xsd#htfl_CommonStockWarrantInitialTermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_d814feed-7218-401e-93d8-4cd287f6d630" xlink:to="loc_htfl_CommonStockWarrantInitialTermLoanMember_a5ed15f4-ca0c-48f4-9bf5-4a60dbd6a7c2" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockWarrantNewMoneyTermLoanMember_7cae010b-a8b9-4abf-ae8b-3a52e395d79d" xlink:href="htfl-20250930.xsd#htfl_CommonStockWarrantNewMoneyTermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_d814feed-7218-401e-93d8-4cd287f6d630" xlink:to="loc_htfl_CommonStockWarrantNewMoneyTermLoanMember_7cae010b-a8b9-4abf-ae8b-3a52e395d79d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockWarrantCreditAgreementMember_a3bfc932-4434-4be2-8d6a-792587575b94" xlink:href="htfl-20250930.xsd#htfl_CommonStockWarrantCreditAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_d814feed-7218-401e-93d8-4cd287f6d630" xlink:to="loc_htfl_CommonStockWarrantCreditAgreementMember_a3bfc932-4434-4be2-8d6a-792587575b94" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_dabbf5f4-41af-4ac8-8c53-c3cc9740b579" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_dabbf5f4-41af-4ac8-8c53-c3cc9740b579" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_f0166039-5f47-46d1-8aa4-d7e40a01a596" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_f0166039-5f47-46d1-8aa4-d7e40a01a596" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstandingTerm_1e4cd22f-f53a-4e74-b24f-63ab40a80b12" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantsAndRightsOutstandingTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_WarrantsAndRightsOutstandingTerm_1e4cd22f-f53a-4e74-b24f-63ab40a80b12" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstanding_4c6c577e-163e-4330-9a5d-8020cfe0eff7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantsAndRightsOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_WarrantsAndRightsOutstanding_4c6c577e-163e-4330-9a5d-8020cfe0eff7" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAdjustmentOfWarrants_a95ca329-fe2b-4ce0-9d8b-cc46a1a7b8f0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAdjustmentOfWarrants"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_f6e1b2d6-1de7-49cf-acbf-440710f4ea51" xlink:to="loc_us-gaap_FairValueAdjustmentOfWarrants_a95ca329-fe2b-4ce0-9d8b-cc46a1a7b8f0" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/CommonStockWarrantLiabilityScheduleofFairValueofCommonStockWarrantLiabilityDeterminedUsingBlackScholesOptionPricingModelDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#CommonStockWarrantLiabilityScheduleofFairValueofCommonStockWarrantLiabilityDeterminedUsingBlackScholesOptionPricingModelDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/CommonStockWarrantLiabilityScheduleofFairValueofCommonStockWarrantLiabilityDeterminedUsingBlackScholesOptionPricingModelDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_bd4ec29a-56d2-44a5-ba21-718776a44933" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_bbf2aef0-535c-4f5b-b755-6a2a0701de4f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_bd4ec29a-56d2-44a5-ba21-718776a44933" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_bbf2aef0-535c-4f5b-b755-6a2a0701de4f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_73ed79f0-b2e1-41e1-b23e-42217e33d09a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_bbf2aef0-535c-4f5b-b755-6a2a0701de4f" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_73ed79f0-b2e1-41e1-b23e-42217e33d09a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_73ed79f0-b2e1-41e1-b23e-42217e33d09a_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_73ed79f0-b2e1-41e1-b23e-42217e33d09a" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_73ed79f0-b2e1-41e1-b23e-42217e33d09a_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_5734bd04-c769-4ee1-ae0c-2261d9228e29" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_73ed79f0-b2e1-41e1-b23e-42217e33d09a" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_5734bd04-c769-4ee1-ae0c-2261d9228e29" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockWarrantLiabilityMember_2a207af1-931d-4b2b-919b-270c10d36fa7" xlink:href="htfl-20250930.xsd#htfl_CommonStockWarrantLiabilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_5734bd04-c769-4ee1-ae0c-2261d9228e29" xlink:to="loc_htfl_CommonStockWarrantLiabilityMember_2a207af1-931d-4b2b-919b-270c10d36fa7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeAxis_efc34bd6-6c70-422d-9131-cea03b14d694" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_bbf2aef0-535c-4f5b-b755-6a2a0701de4f" xlink:to="loc_us-gaap_MeasurementInputTypeAxis_efc34bd6-6c70-422d-9131-cea03b14d694" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeDomain_efc34bd6-6c70-422d-9131-cea03b14d694_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_MeasurementInputTypeAxis_efc34bd6-6c70-422d-9131-cea03b14d694" xlink:to="loc_us-gaap_MeasurementInputTypeDomain_efc34bd6-6c70-422d-9131-cea03b14d694_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_MeasurementInputTypeAxis_efc34bd6-6c70-422d-9131-cea03b14d694" xlink:to="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputSharePriceMember_59a67ad8-3ec9-4eb0-83af-fe1b30a623b7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputSharePriceMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputSharePriceMember_59a67ad8-3ec9-4eb0-83af-fe1b30a623b7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputExercisePriceMember_7143ad09-1950-4bb0-8136-a9bed53486b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputExercisePriceMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputExercisePriceMember_7143ad09-1950-4bb0-8136-a9bed53486b9" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputExpectedTermMember_cccd187e-7eaf-43f8-8681-b2f92b977999" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputExpectedTermMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputExpectedTermMember_cccd187e-7eaf-43f8-8681-b2f92b977999" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputPriceVolatilityMember_e4ce4c8c-4d99-4ff6-afdc-e029ab5543e6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputPriceVolatilityMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputPriceVolatilityMember_e4ce4c8c-4d99-4ff6-afdc-e029ab5543e6" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputRiskFreeInterestRateMember_37e3894c-ebdf-4ea5-9b14-58146325f207" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputRiskFreeInterestRateMember_37e3894c-ebdf-4ea5-9b14-58146325f207" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputExpectedDividendRateMember_0b489e89-b5b1-495b-84fc-1e9c2f9df8b4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputExpectedDividendRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_65b270da-3a90-43c7-a04a-7f201a6f35f8" xlink:to="loc_us-gaap_MeasurementInputExpectedDividendRateMember_0b489e89-b5b1-495b-84fc-1e9c2f9df8b4" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstandingMeasurementInput_4d88ac8c-9a6a-482a-b623-17305b6b2800" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantsAndRightsOutstandingMeasurementInput"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_bd4ec29a-56d2-44a5-ba21-718776a44933" xlink:to="loc_us-gaap_WarrantsAndRightsOutstandingMeasurementInput_4d88ac8c-9a6a-482a-b623-17305b6b2800" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/DerivativeLiabilityNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#DerivativeLiabilityNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/DerivativeLiabilityNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_371b3d09-7515-4438-bcb0-670cf558d6ef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_0656f65d-27ce-43e8-81dc-a8116fde341d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_371b3d09-7515-4438-bcb0-670cf558d6ef" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_0656f65d-27ce-43e8-81dc-a8116fde341d" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_0656f65d-27ce-43e8-81dc-a8116fde341d" xlink:to="loc_us-gaap_DebtInstrumentAxis_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_b01e4ab2-b1db-4c34-85d1-4bd33cfb3c22" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_7c7b6cf0-1bd3-408b-b9cc-2b7531657e82" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_b01e4ab2-b1db-4c34-85d1-4bd33cfb3c22" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2024TermLoanMember_10def124-265f-4011-8405-963c5315e48e" xlink:href="htfl-20250930.xsd#htfl_A2024TermLoanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_b01e4ab2-b1db-4c34-85d1-4bd33cfb3c22" xlink:to="loc_htfl_A2024TermLoanMember_10def124-265f-4011-8405-963c5315e48e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025ConvertiblePromissoryNotesMember_16fbf73b-21d8-41f4-9b48-fc2cebbe19bb" xlink:href="htfl-20250930.xsd#htfl_A2025ConvertiblePromissoryNotesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_b01e4ab2-b1db-4c34-85d1-4bd33cfb3c22" xlink:to="loc_htfl_A2025ConvertiblePromissoryNotesMember_16fbf73b-21d8-41f4-9b48-fc2cebbe19bb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeAxis_53414793-1532-470d-bc81-a9c908251a1f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_0656f65d-27ce-43e8-81dc-a8116fde341d" xlink:to="loc_us-gaap_LongtermDebtTypeAxis_53414793-1532-470d-bc81-a9c908251a1f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_53414793-1532-470d-bc81-a9c908251a1f_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_53414793-1532-470d-bc81-a9c908251a1f" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_53414793-1532-470d-bc81-a9c908251a1f_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LongtermDebtTypeDomain_9ce04553-e43a-498c-839b-b778069cfba2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermDebtTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_LongtermDebtTypeAxis_53414793-1532-470d-bc81-a9c908251a1f" xlink:to="loc_us-gaap_LongtermDebtTypeDomain_9ce04553-e43a-498c-839b-b778069cfba2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ConvertibleDebtMember_de045bc8-abeb-4ad3-895f-939bd1425c8a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConvertibleDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_LongtermDebtTypeDomain_9ce04553-e43a-498c-839b-b778069cfba2" xlink:to="loc_us-gaap_ConvertibleDebtMember_de045bc8-abeb-4ad3-895f-939bd1425c8a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_UnrealizedGainLossOnDerivatives_b4b1b46a-ce07-4bea-985c-4867c2fa3bb9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_UnrealizedGainLossOnDerivatives"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_371b3d09-7515-4438-bcb0-670cf558d6ef" xlink:to="loc_us-gaap_UnrealizedGainLossOnDerivatives_b4b1b46a-ce07-4bea-985c-4867c2fa3bb9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilities_3e9de72f-66c8-4179-baa1-b5fdb3efd15d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_371b3d09-7515-4438-bcb0-670cf558d6ef" xlink:to="loc_us-gaap_DerivativeLiabilities_3e9de72f-66c8-4179-baa1-b5fdb3efd15d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_DebtInstrumentConversionPriceDiscountPercentage_ce122a1e-21a0-41ef-9712-406ea773f528" xlink:href="htfl-20250930.xsd#htfl_DebtInstrumentConversionPriceDiscountPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_371b3d09-7515-4438-bcb0-670cf558d6ef" xlink:to="loc_htfl_DebtInstrumentConversionPriceDiscountPercentage_ce122a1e-21a0-41ef-9712-406ea773f528" xlink:type="arc" order="2"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/DerivativeLiabilityScheduleofDerivativeInstrumentsDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#DerivativeLiabilityScheduleofDerivativeInstrumentsDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/DerivativeLiabilityScheduleofDerivativeInstrumentsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_08e349de-ddc6-4f8f-8fd6-92e72da46831" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_ac4a92a7-1438-463f-a688-9839b7fd7616" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_08e349de-ddc6-4f8f-8fd6-92e72da46831" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_ac4a92a7-1438-463f-a688-9839b7fd7616" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeAxis_9e322b6c-d981-444e-9c4f-a55d2f431322" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_ac4a92a7-1438-463f-a688-9839b7fd7616" xlink:to="loc_us-gaap_MeasurementInputTypeAxis_9e322b6c-d981-444e-9c4f-a55d2f431322" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeDomain_9e322b6c-d981-444e-9c4f-a55d2f431322_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_MeasurementInputTypeAxis_9e322b6c-d981-444e-9c4f-a55d2f431322" xlink:to="loc_us-gaap_MeasurementInputTypeDomain_9e322b6c-d981-444e-9c4f-a55d2f431322_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_MeasurementInputTypeAxis_9e322b6c-d981-444e-9c4f-a55d2f431322" xlink:to="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_MeasurementInputDebtYieldMember_94116661-f8e5-49cc-9bcb-a2a06f502afa" xlink:href="htfl-20250930.xsd#htfl_MeasurementInputDebtYieldMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_htfl_MeasurementInputDebtYieldMember_94116661-f8e5-49cc-9bcb-a2a06f502afa" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember_9b8183fc-351f-4d53-ab29-8370c6b50f33" xlink:href="htfl-20250930.xsd#htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember_9b8183fc-351f-4d53-ab29-8370c6b50f33" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputDefaultRateMember_2fe64678-9077-4169-88f1-a40ca4a682ef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputDefaultRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_us-gaap_MeasurementInputDefaultRateMember_2fe64678-9077-4169-88f1-a40ca4a682ef" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_MeasurementInputProbabilityToIncurNewDebtMember_f282056a-fe54-4091-92e3-6bd1f8c756ae" xlink:href="htfl-20250930.xsd#htfl_MeasurementInputProbabilityToIncurNewDebtMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_htfl_MeasurementInputProbabilityToIncurNewDebtMember_f282056a-fe54-4091-92e3-6bd1f8c756ae" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_MeasurementInputProbabilityOfChangeOfControlMember_f3591956-0975-4a8b-9dcd-b07c77ad625f" xlink:href="htfl-20250930.xsd#htfl_MeasurementInputProbabilityOfChangeOfControlMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_htfl_MeasurementInputProbabilityOfChangeOfControlMember_f3591956-0975-4a8b-9dcd-b07c77ad625f" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MeasurementInputDiscountRateMember_7a64126e-7344-4ba7-bf9d-fe938cf886f6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputDiscountRateMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_MeasurementInputTypeDomain_70942bd5-d612-4e53-868d-3c4b31ee5095" xlink:to="loc_us-gaap_MeasurementInputDiscountRateMember_7a64126e-7344-4ba7-bf9d-fe938cf886f6" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ValuationTechniqueAxis_6862fe13-c33c-4d3b-8ff0-22ef019b8e75" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable_ac4a92a7-1438-463f-a688-9839b7fd7616" xlink:to="loc_us-gaap_ValuationTechniqueAxis_6862fe13-c33c-4d3b-8ff0-22ef019b8e75" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ValuationTechniqueDomain_6862fe13-c33c-4d3b-8ff0-22ef019b8e75_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ValuationTechniqueAxis_6862fe13-c33c-4d3b-8ff0-22ef019b8e75" xlink:to="loc_us-gaap_ValuationTechniqueDomain_6862fe13-c33c-4d3b-8ff0-22ef019b8e75_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ValuationTechniqueDomain_4b3b8e5f-85f3-4c83-a66c-bb9465ebf4e6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ValuationTechniqueAxis_6862fe13-c33c-4d3b-8ff0-22ef019b8e75" xlink:to="loc_us-gaap_ValuationTechniqueDomain_4b3b8e5f-85f3-4c83-a66c-bb9465ebf4e6" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_BlackScholesMertonModelMember_f829864a-bf5f-4f37-83eb-e7acfc86cb18" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BlackScholesMertonModelMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ValuationTechniqueDomain_4b3b8e5f-85f3-4c83-a66c-bb9465ebf4e6" xlink:to="loc_us-gaap_BlackScholesMertonModelMember_f829864a-bf5f-4f37-83eb-e7acfc86cb18" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_MonteCarloModelMember_9bccad28-f59c-41d0-9ec2-61598ce4851f" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MonteCarloModelMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ValuationTechniqueDomain_4b3b8e5f-85f3-4c83-a66c-bb9465ebf4e6" xlink:to="loc_us-gaap_MonteCarloModelMember_9bccad28-f59c-41d0-9ec2-61598ce4851f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DerivativeLiabilityMeasurementInput_499a22b5-f092-4bc7-b473-a708b0486ac8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilityMeasurementInput"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems_08e349de-ddc6-4f8f-8fd6-92e72da46831" xlink:to="loc_us-gaap_DerivativeLiabilityMeasurementInput_499a22b5-f092-4bc7-b473-a708b0486ac8" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/StockBasedCompensationPlansNarrativeDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#StockBasedCompensationPlansNarrativeDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/StockBasedCompensationPlansNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:to="loc_us-gaap_AwardTypeAxis_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_02d5f283-75b8-4e81-b154-af9a2c626438" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_9dd8f3b5-9df9-489a-9cf9-d96f88b447fe" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_02d5f283-75b8-4e81-b154-af9a2c626438" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_030092ed-282f-4b83-b32f-ab4d8005dc3a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_02d5f283-75b8-4e81-b154-af9a2c626438" xlink:to="loc_us-gaap_EmployeeStockOptionMember_030092ed-282f-4b83-b32f-ab4d8005dc3a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_0917726b-901c-42d2-ae47-1d6d77532544" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_02d5f283-75b8-4e81-b154-af9a2c626438" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_0917726b-901c-42d2-ae47-1d6d77532544" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_4fa09d87-d13c-409c-bab7-f50e3de1db01" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_02d5f283-75b8-4e81-b154-af9a2c626438" xlink:to="loc_us-gaap_EmployeeStockMember_4fa09d87-d13c-409c-bab7-f50e3de1db01" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusAxis_daa678c5-c89e-4c8c-9fa1-9100309d5eb8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:to="loc_us-gaap_GranteeStatusAxis_daa678c5-c89e-4c8c-9fa1-9100309d5eb8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusDomain_daa678c5-c89e-4c8c-9fa1-9100309d5eb8_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_GranteeStatusAxis_daa678c5-c89e-4c8c-9fa1-9100309d5eb8" xlink:to="loc_us-gaap_GranteeStatusDomain_daa678c5-c89e-4c8c-9fa1-9100309d5eb8_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusDomain_9c6ee2ce-2ff5-495c-bd2c-93d4372d4159" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GranteeStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_GranteeStatusAxis_daa678c5-c89e-4c8c-9fa1-9100309d5eb8" xlink:to="loc_us-gaap_GranteeStatusDomain_9c6ee2ce-2ff5-495c-bd2c-93d4372d4159" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedPaymentArrangementEmployeeMember_2736d554-3d50-4345-a4e1-2c682acdf88c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GranteeStatusDomain_9c6ee2ce-2ff5-495c-bd2c-93d4372d4159" xlink:to="loc_us-gaap_ShareBasedPaymentArrangementEmployeeMember_2736d554-3d50-4345-a4e1-2c682acdf88c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingAxis_78b94694-b8a2-44d7-afd3-e926c37721da" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:to="loc_us-gaap_VestingAxis_78b94694-b8a2-44d7-afd3-e926c37721da" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingDomain_78b94694-b8a2-44d7-afd3-e926c37721da_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_VestingAxis_78b94694-b8a2-44d7-afd3-e926c37721da" xlink:to="loc_us-gaap_VestingDomain_78b94694-b8a2-44d7-afd3-e926c37721da_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_VestingDomain_20788a7f-2ac2-455e-b605-7ef7c3562c3c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_VestingAxis_78b94694-b8a2-44d7-afd3-e926c37721da" xlink:to="loc_us-gaap_VestingDomain_20788a7f-2ac2-455e-b605-7ef7c3562c3c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationAwardTrancheOneMember_e2f8de55-69de-49c0-bcb4-6c1f34457438" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VestingDomain_20788a7f-2ac2-455e-b605-7ef7c3562c3c" xlink:to="loc_us-gaap_ShareBasedCompensationAwardTrancheOneMember_e2f8de55-69de-49c0-bcb4-6c1f34457438" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationAwardTrancheTwoMember_fb66a665-e3d2-4604-890d-835b2eb7ca44" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationAwardTrancheTwoMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_VestingDomain_20788a7f-2ac2-455e-b605-7ef7c3562c3c" xlink:to="loc_us-gaap_ShareBasedCompensationAwardTrancheTwoMember_fb66a665-e3d2-4604-890d-835b2eb7ca44" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_e0ebdb09-5175-42a2-b9c0-e37578460493" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_73b7b2d4-5faa-4456-a306-eb6e25fea81a" xlink:to="loc_us-gaap_PlanNameAxis_e0ebdb09-5175-42a2-b9c0-e37578460493" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_e0ebdb09-5175-42a2-b9c0-e37578460493_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_e0ebdb09-5175-42a2-b9c0-e37578460493" xlink:to="loc_us-gaap_PlanNameDomain_e0ebdb09-5175-42a2-b9c0-e37578460493_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_b4f37228-3b2f-4990-adca-c8b1a9b60356" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_e0ebdb09-5175-42a2-b9c0-e37578460493" xlink:to="loc_us-gaap_PlanNameDomain_b4f37228-3b2f-4990-adca-c8b1a9b60356" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025PerformanceIncentivePlanMember_ef451c23-860e-45d0-a312-c78e9ae2cd66" xlink:href="htfl-20250930.xsd#htfl_A2025PerformanceIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_b4f37228-3b2f-4990-adca-c8b1a9b60356" xlink:to="loc_htfl_A2025PerformanceIncentivePlanMember_ef451c23-860e-45d0-a312-c78e9ae2cd66" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_A2025EmployeeStockPurchasePlanMember_92a23d3a-6cb4-4647-b75a-dbe5ef1e5d76" xlink:href="htfl-20250930.xsd#htfl_A2025EmployeeStockPurchasePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_b4f37228-3b2f-4990-adca-c8b1a9b60356" xlink:to="loc_htfl_A2025EmployeeStockPurchasePlanMember_92a23d3a-6cb4-4647-b75a-dbe5ef1e5d76" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_0ddf5c9a-7fec-493f-a8c9-603b0a0bac2d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_CommonStockCapitalSharesReservedForFutureIssuance_0ddf5c9a-7fec-493f-a8c9-603b0a0bac2d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CommonStockOutstandingPercentage_a9f13d6b-b356-461f-872a-ca86a8b573e3" xlink:href="htfl-20250930.xsd#htfl_CommonStockOutstandingPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_htfl_CommonStockOutstandingPercentage_a9f13d6b-b356-461f-872a-ca86a8b573e3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_27ab0a7c-2b93-43b6-8143-ee5697f12ebe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod_27ab0a7c-2b93-43b6-8143-ee5697f12ebe" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_583bea0e-11ee-409f-a401-4a4be8f6956a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent_583bea0e-11ee-409f-a401-4a4be8f6956a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_PercentageOfVotingPowerOnClassesOfStock_27432ad4-65cd-4943-8b82-fdc1c900d7d1" xlink:href="htfl-20250930.xsd#htfl_PercentageOfVotingPowerOnClassesOfStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_htfl_PercentageOfVotingPowerOnClassesOfStock_27432ad4-65cd-4943-8b82-fdc1c900d7d1" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_9dd0375d-0f0c-4611-8062-d1a292da24ff" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1_9dd0375d-0f0c-4611-8062-d1a292da24ff" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage_c5f82029-ba89-46cd-8833-2bb20ae0d3e9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage_c5f82029-ba89-46cd-8833-2bb20ae0d3e9" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_7cb2ba01-503b-41b6-91cd-80a4fa9c2b19" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_7cb2ba01-503b-41b6-91cd-80a4fa9c2b19" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue_4dcf9af5-5f53-4064-a96e-f01bea52e725" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue_4dcf9af5-5f53-4064-a96e-f01bea52e725" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_fab3e990-f5b2-450e-a421-0b19c53b9a22" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_fab3e990-f5b2-450e-a421-0b19c53b9a22" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_185ddff0-f16a-4824-8775-5b6934483eb2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_185ddff0-f16a-4824-8775-5b6934483eb2" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_0f98eaad-921b-487f-8e1e-beb2dfe3c1b8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_53f9fd24-b674-4b4e-b653-97feaa37ec7e" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions_0f98eaad-921b-487f-8e1e-beb2dfe3c1b8" xlink:type="arc" order="11"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/StockBasedCompensationPlansScheduleofRestrictedStockUnitActivityDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#StockBasedCompensationPlansScheduleofRestrictedStockUnitActivityDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/StockBasedCompensationPlansScheduleofRestrictedStockUnitActivityDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_e91fc286-1c08-40aa-b684-2157b0942150" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_1b764642-6bf8-48c3-a55d-9aa04b5fce85" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_e91fc286-1c08-40aa-b684-2157b0942150" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_1b764642-6bf8-48c3-a55d-9aa04b5fce85" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_12d621b0-1f77-4eba-8f29-ef257a97d487" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_1b764642-6bf8-48c3-a55d-9aa04b5fce85" xlink:to="loc_us-gaap_AwardTypeAxis_12d621b0-1f77-4eba-8f29-ef257a97d487" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_12d621b0-1f77-4eba-8f29-ef257a97d487_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_12d621b0-1f77-4eba-8f29-ef257a97d487" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_12d621b0-1f77-4eba-8f29-ef257a97d487_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_459c018d-c470-4edb-8bd1-92a30de3db00" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_12d621b0-1f77-4eba-8f29-ef257a97d487" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_459c018d-c470-4edb-8bd1-92a30de3db00" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_15dd11f0-a849-41c6-8d6e-02d199978501" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_459c018d-c470-4edb-8bd1-92a30de3db00" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_15dd11f0-a849-41c6-8d6e-02d199978501" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_e91fc286-1c08-40aa-b684-2157b0942150" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_fd07f619-a4d3-4ac6-aa83-38f7d05c76e8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_fd07f619-a4d3-4ac6-aa83-38f7d05c76e8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_b8554c4c-86ae-483a-96cf-9a8e12ab91c6" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod_b8554c4c-86ae-483a-96cf-9a8e12ab91c6" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_d2c253c5-79c0-4f3b-8a44-dfea034d1506" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod_d2c253c5-79c0-4f3b-8a44-dfea034d1506" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_5db1806e-070f-488e-84c3-a878da3626a0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward_55c684d9-c4b6-4183-ae1e-b433f0f372b0" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod_5db1806e-070f-488e-84c3-a878da3626a0" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber_ac0b818e-68ca-4228-b5d0-c9bc8f7c740b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_e91fc286-1c08-40aa-b684-2157b0942150" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_9026e8b3-54b0-4972-b152-71ab40503ee0" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_9026e8b3-54b0-4972-b152-71ab40503ee0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_57b33e83-bd55-4e7b-848b-c45cdf4f9b82" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_57b33e83-bd55-4e7b-848b-c45cdf4f9b82" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_de622b6c-628f-4d80-92f5-d0c1bc283fe9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue_de622b6c-628f-4d80-92f5-d0c1bc283fe9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_0909aab3-e6ef-4039-a2fe-58d678e24c68" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward_a942dafb-b20d-4fcc-88d2-26baed5899cf" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue_0909aab3-e6ef-4039-a2fe-58d678e24c68" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue_4e6ea29f-acce-40b4-9fd2-cbaa18ad5d19" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/StockBasedCompensationPlansScheduleofFairValueOfStockOptionsUsingTheBlackScholesOptionPricingModelDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#StockBasedCompensationPlansScheduleofFairValueOfStockOptionsUsingTheBlackScholesOptionPricingModelDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/StockBasedCompensationPlansScheduleofFairValueOfStockOptionsUsingTheBlackScholesOptionPricingModelDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_40319744-7a52-4ff0-b7a7-bf9e640f0d4a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_40319744-7a52-4ff0-b7a7-bf9e640f0d4a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_21547016-8133-4854-9e63-f6e4968e1bef" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_40319744-7a52-4ff0-b7a7-bf9e640f0d4a" xlink:to="loc_us-gaap_AwardTypeAxis_21547016-8133-4854-9e63-f6e4968e1bef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_21547016-8133-4854-9e63-f6e4968e1bef_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_21547016-8133-4854-9e63-f6e4968e1bef" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_21547016-8133-4854-9e63-f6e4968e1bef_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_7f3a213c-4ac3-4ec6-a430-2d9e0f536cfe" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_21547016-8133-4854-9e63-f6e4968e1bef" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_7f3a213c-4ac3-4ec6-a430-2d9e0f536cfe" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_f99e33b2-d24c-4e30-85c9-2c1ce2d1546e" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_7f3a213c-4ac3-4ec6-a430-2d9e0f536cfe" xlink:to="loc_us-gaap_EmployeeStockOptionMember_f99e33b2-d24c-4e30-85c9-2c1ce2d1546e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_f745819a-06c5-484a-8186-37e3e0d0d587" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_7f3a213c-4ac3-4ec6-a430-2d9e0f536cfe" xlink:to="loc_us-gaap_EmployeeStockMember_f745819a-06c5-484a-8186-37e3e0d0d587" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_cfb0b045-5b28-4450-83a4-a31d635e284a" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_cfb0b045-5b28-4450-83a4-a31d635e284a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum_9287f5d0-1889-4c9a-a6fb-ef22dce44b27" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum_9287f5d0-1889-4c9a-a6fb-ef22dce44b27" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum_03a6443b-c2e2-47a7-9096-52f37a18b675" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum_03a6443b-c2e2-47a7-9096-52f37a18b675" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_c9304a30-0d27-41f3-a9f1-0f70d7af6f8d" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate_c9304a30-0d27-41f3-a9f1-0f70d7af6f8d" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum_72f59ba3-ff26-42c3-8579-dfc2cf509e28" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMinimum_72f59ba3-ff26-42c3-8579-dfc2cf509e28" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum_a52140c7-511c-4aa8-9b67-520fea246402" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRateMaximum_a52140c7-511c-4aa8-9b67-520fea246402" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_5ecec35d-b01a-419e-a2cb-fa1549b6f1cc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_5ecec35d-b01a-419e-a2cb-fa1549b6f1cc" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_44f1fbf2-58a4-42c7-a742-1c2fb93840be" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_678890b4-e0d5-4965-b027-7e768c8ba470" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_44f1fbf2-58a4-42c7-a742-1c2fb93840be" xlink:type="arc" order="7"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/StockBasedCompensationPlansScheduleofStockBasedCompensationExpenseDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#StockBasedCompensationPlansScheduleofStockBasedCompensationExpenseDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/StockBasedCompensationPlansScheduleofStockBasedCompensationExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_bcab9e36-620c-4edd-9cf4-960326ca0e90" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_6acdb415-3a61-4c6d-b87b-b80d1f9329ac" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_bcab9e36-620c-4edd-9cf4-960326ca0e90" xlink:to="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_6acdb415-3a61-4c6d-b87b-b80d1f9329ac" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationAxis_f8604961-0d9f-41e3-9358-758624f98449" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_6acdb415-3a61-4c6d-b87b-b80d1f9329ac" xlink:to="loc_us-gaap_IncomeStatementLocationAxis_f8604961-0d9f-41e3-9358-758624f98449" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_f8604961-0d9f-41e3-9358-758624f98449_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_f8604961-0d9f-41e3-9358-758624f98449" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_f8604961-0d9f-41e3-9358-758624f98449_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncomeStatementLocationDomain_f225da02-5259-4696-89dc-c428b0595233" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_IncomeStatementLocationAxis_f8604961-0d9f-41e3-9358-758624f98449" xlink:to="loc_us-gaap_IncomeStatementLocationDomain_f225da02-5259-4696-89dc-c428b0595233" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CostOfSalesMember_124e5aec-8d40-4b18-a3bf-8f0906c9d68b" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfSalesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_f225da02-5259-4696-89dc-c428b0595233" xlink:to="loc_us-gaap_CostOfSalesMember_124e5aec-8d40-4b18-a3bf-8f0906c9d68b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ResearchAndDevelopmentExpenseMember_2b5cf781-534b-4aac-afe3-f56d5e73e0ae" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_f225da02-5259-4696-89dc-c428b0595233" xlink:to="loc_us-gaap_ResearchAndDevelopmentExpenseMember_2b5cf781-534b-4aac-afe3-f56d5e73e0ae" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GeneralAndAdministrativeExpenseMember_e8e45276-3edb-43ef-ad9b-2184b0793272" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncomeStatementLocationDomain_f225da02-5259-4696-89dc-c428b0595233" xlink:to="loc_us-gaap_GeneralAndAdministrativeExpenseMember_e8e45276-3edb-43ef-ad9b-2184b0793272" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_45368504-b012-4699-b2f7-280f384dc8b9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_bcab9e36-620c-4edd-9cf4-960326ca0e90" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_45368504-b012-4699-b2f7-280f384dc8b9" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/NetLossPerShareScheduleofOutstandingSharesofPotentiallyDilutiveSecuritiesDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#NetLossPerShareScheduleofOutstandingSharesofPotentiallyDilutiveSecuritiesDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/NetLossPerShareScheduleofOutstandingSharesofPotentiallyDilutiveSecuritiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_08d191fc-2701-445f-aa1f-d5f22b9a4dc3" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_3a56300e-7255-443a-9695-c97d44a34c39" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_08d191fc-2701-445f-aa1f-d5f22b9a4dc3" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_3a56300e-7255-443a-9695-c97d44a34c39" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_eedae498-f364-491f-b2da-17dfabbcfbe4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_3a56300e-7255-443a-9695-c97d44a34c39" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_eedae498-f364-491f-b2da-17dfabbcfbe4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_eedae498-f364-491f-b2da-17dfabbcfbe4_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_eedae498-f364-491f-b2da-17dfabbcfbe4" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_eedae498-f364-491f-b2da-17dfabbcfbe4_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_eedae498-f364-491f-b2da-17dfabbcfbe4" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RedeemableConvertiblePreferredStockMember_ddee2203-9ea5-4094-a01f-9fc0db6b3716" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:to="loc_us-gaap_RedeemableConvertiblePreferredStockMember_ddee2203-9ea5-4094-a01f-9fc0db6b3716" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_2e6dfdc0-f3c1-4db1-9e72-daac6c5bc200" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:to="loc_us-gaap_EmployeeStockOptionMember_2e6dfdc0-f3c1-4db1-9e72-daac6c5bc200" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedStockUnitsRSUMember_2ef3c73c-c762-4fda-8e88-54e7823cf0f9" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:to="loc_us-gaap_RestrictedStockUnitsRSUMember_2ef3c73c-c762-4fda-8e88-54e7823cf0f9" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockMember_39155d46-959a-424d-a81c-573accde6c18" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:to="loc_us-gaap_EmployeeStockMember_39155d46-959a-424d-a81c-573accde6c18" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_276e0e51-a673-4508-b861-4218b5a29c96" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_dbd28509-8a8b-4a29-ab44-6cea5ea7abd8" xlink:to="loc_us-gaap_WarrantMember_276e0e51-a673-4508-b861-4218b5a29c96" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_40140e53-ec28-4e67-ad05-785cd972cd76" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_08d191fc-2701-445f-aa1f-d5f22b9a4dc3" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_40140e53-ec28-4e67-ad05-785cd972cd76" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://heartflow.com/role/SubsequentEventsDetails" xlink:type="simple" xlink:href="htfl-20250930.xsd#SubsequentEventsDetails"/>
  <link:definitionLink xlink:role="http://heartflow.com/role/SubsequentEventsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTable_2cd14333-5bd9-4d08-b745-e00b3e0b95a2" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_SubsequentEventTable_2cd14333-5bd9-4d08-b745-e00b3e0b95a2" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementGeographicalAxis_8628a354-0231-4540-9d45-312fa7978cfc" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_2cd14333-5bd9-4d08-b745-e00b3e0b95a2" xlink:to="loc_srt_StatementGeographicalAxis_8628a354-0231-4540-9d45-312fa7978cfc" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_8628a354-0231-4540-9d45-312fa7978cfc_default" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementGeographicalAxis_8628a354-0231-4540-9d45-312fa7978cfc" xlink:to="loc_srt_SegmentGeographicalDomain_8628a354-0231-4540-9d45-312fa7978cfc_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_SegmentGeographicalDomain_32f4562b-f798-4b51-bf4e-6eb4596afe05" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementGeographicalAxis_8628a354-0231-4540-9d45-312fa7978cfc" xlink:to="loc_srt_SegmentGeographicalDomain_32f4562b-f798-4b51-bf4e-6eb4596afe05" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_SanFranciscoCaliforniaMember_3ce4d420-e96c-4245-8872-49e6ff204666" xlink:href="htfl-20250930.xsd#htfl_SanFranciscoCaliforniaMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_SegmentGeographicalDomain_32f4562b-f798-4b51-bf4e-6eb4596afe05" xlink:to="loc_htfl_SanFranciscoCaliforniaMember_3ce4d420-e96c-4245-8872-49e6ff204666" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_75285886-77a5-48a4-9406-2071561da7ed" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_2cd14333-5bd9-4d08-b745-e00b3e0b95a2" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_75285886-77a5-48a4-9406-2071561da7ed" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_75285886-77a5-48a4-9406-2071561da7ed_default" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_75285886-77a5-48a4-9406-2071561da7ed" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_75285886-77a5-48a4-9406-2071561da7ed_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_fa08ab57-ca56-49a3-a595-8062b9d01337" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_75285886-77a5-48a4-9406-2071561da7ed" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_fa08ab57-ca56-49a3-a595-8062b9d01337" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_5bf3d621-d2af-46a7-b36f-f72678e126a4" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_fa08ab57-ca56-49a3-a595-8062b9d01337" xlink:to="loc_us-gaap_SubsequentEventMember_5bf3d621-d2af-46a7-b36f-f72678e126a4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetRentableArea_b18bddae-c6fc-4611-a8ed-0d1c6b61c824" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetRentableArea"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_NetRentableArea_b18bddae-c6fc-4611-a8ed-0d1c6b61c824" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseTermOfContract_431a582f-e6e5-4309-a1b2-66ead6ea26b7" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseTermOfContract"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_LesseeOperatingLeaseTermOfContract_431a582f-e6e5-4309-a1b2-66ead6ea26b7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_LesseeOperatingLeaseNumberOfRenewalOptions_0254d77a-769e-44a2-a13c-dbe5cee1dd83" xlink:href="htfl-20250930.xsd#htfl_LesseeOperatingLeaseNumberOfRenewalOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_htfl_LesseeOperatingLeaseNumberOfRenewalOptions_0254d77a-769e-44a2-a13c-dbe5cee1dd83" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LesseeOperatingLeaseRenewalTerm_9f04c0ed-a153-479c-b9c9-49b975e113e5" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_LesseeOperatingLeaseRenewalTerm_9f04c0ed-a153-479c-b9c9-49b975e113e5" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SecurityDeposit_ce694b39-8bf1-422e-b1c0-07bac3707f93" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SecurityDeposit"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_SecurityDeposit_ce694b39-8bf1-422e-b1c0-07bac3707f93" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_LesseeOperatingLeaseAverageMonthlyPayment_e03fb483-b66b-4e3a-abde-6c5813fd7f1f" xlink:href="htfl-20250930.xsd#htfl_LesseeOperatingLeaseAverageMonthlyPayment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_htfl_LesseeOperatingLeaseAverageMonthlyPayment_e03fb483-b66b-4e3a-abde-6c5813fd7f1f" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_07fce142-23c7-4219-9e48-56277b247a5c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_07fce142-23c7-4219-9e48-56277b247a5c" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAdjustmentOfWarrants_5ef2d1c9-1170-47f1-b2de-c282bd4ced11" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAdjustmentOfWarrants"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_FairValueAdjustmentOfWarrants_5ef2d1c9-1170-47f1-b2de-c282bd4ced11" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantsAndRightsOutstanding_32eec6a1-2d18-4a60-ab97-a0b9cc1a319c" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantsAndRightsOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_26817285-0290-4897-aed4-9a16e13bf7fc" xlink:to="loc_us-gaap_WarrantsAndRightsOutstanding_32eec6a1-2d18-4a60-ab97-a0b9cc1a319c" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:type="simple" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#IndividualsOnly"/>
  <link:definitionLink xlink:role="http://xbrl.sec.gov/ecd/role/IndividualsOnly" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_ecd_AllIndividualsMember" xlink:href="https://xbrl.sec.gov/ecd/2025/ecd-2025.xsd#ecd_AllIndividualsMember"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_JohnC.M.FarquharMember_3f1fe1c5-2d49-489b-be2f-b371368b963f" xlink:href="htfl-20250930.xsd#htfl_JohnC.M.FarquharMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_ecd_AllIndividualsMember" xlink:to="loc_htfl_JohnC.M.FarquharMember_3f1fe1c5-2d49-489b-be2f-b371368b963f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_CampbellD.K.RogersMember_619bced0-5bbb-40b4-b6e2-9e2aaaed9f9a" xlink:href="htfl-20250930.xsd#htfl_CampbellD.K.RogersMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_ecd_AllIndividualsMember" xlink:to="loc_htfl_CampbellD.K.RogersMember_619bced0-5bbb-40b4-b6e2-9e2aaaed9f9a" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_htfl_JulieA.CullivanMember_f95a5281-6d6b-4b52-8d6d-1ecb0464e2fa" xlink:href="htfl-20250930.xsd#htfl_JulieA.CullivanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_ecd_AllIndividualsMember" xlink:to="loc_htfl_JulieA.CullivanMember_f95a5281-6d6b-4b52-8d6d-1ecb0464e2fa" xlink:type="arc" order="4"/>
  </link:definitionLink>
</link:linkbase>
