<?xml version="1.0"?>
<ownershipDocument>

    <schemaVersion>X0508</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2025-05-30</periodOfReport>

    <issuer>
        <issuerCik>0001865494</issuerCik>
        <issuerName>IO Biotech, Inc.</issuerName>
        <issuerTradingSymbol>IOBT</issuerTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001891841</rptOwnerCik>
            <rptOwnerName>Hunter Heidi</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>C/O IO BIOTECH, INC.</rptOwnerStreet1>
            <rptOwnerStreet2>OLE MAALOES VEJ 3</rptOwnerStreet2>
            <rptOwnerCity>COPENHAGEN</rptOwnerCity>
            <rptOwnerState>G7</rptOwnerState>
            <rptOwnerZipCode>2200</rptOwnerZipCode>
            <rptOwnerStateDescription>DENMARK</rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>true</isDirector>
        </reportingOwnerRelationship>
    </reportingOwner>

    <aff10b5One>false</aff10b5One>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2025-05-30</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>P</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>15000</value>
                </transactionShares>
                <transactionPricePerShare>
                    <value>1.3923</value>
                    <footnoteId id="F1"/>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>A</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>15000</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
    </nonDerivativeTable>

    <footnotes>
        <footnote id="F1">The transaction was executed in multiple trades in prices ranging from $1.37 to $1.40, inclusive. The price reported in Column 4 above reflects the weighted average purchase price. The reporting person hereby undertakes to provide to the Securities and Exchange Commission staff, the Issuer, or a security holder of the Issuer, upon request, full information regarding the number of shares purchased at each separate price within the range set forth in this footnote.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>/s/ Brian Burkavage, Attorney-in-Fact for Heidi Hunter</signatureName>
        <signatureDate>2025-06-02</signatureDate>
    </ownerSignature>
</ownershipDocument>
