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

    <schemaVersion>X0707</schemaVersion>

    <submissionType>D/A</submissionType>

    <testOrLive>LIVE</testOrLive>

    <primaryIssuer>
        <cik>0001554907</cik>
        <entityName>Tolero Pharmaceuticals, Inc.</entityName>
        <issuerAddress>
            <street1>2975 Executive Parkway</street1>
            <street2>Suite 320</street2>
            <city>Lehi</city>
            <stateOrCountry>UT</stateOrCountry>
            <stateOrCountryDescription>UTAH</stateOrCountryDescription>
            <zipCode>84043</zipCode>
        </issuerAddress>
        <issuerPhoneNumber>801-285-6003</issuerPhoneNumber>
        <jurisdictionOfInc>DELAWARE</jurisdictionOfInc>
        <issuerPreviousNameList>
            <value>None</value>
        </issuerPreviousNameList>
        <edgarPreviousNameList>
            <value>None</value>
        </edgarPreviousNameList>
        <entityType>Corporation</entityType>
        <yearOfInc>
            <withinFiveYears>true</withinFiveYears>
            <value>2011</value>
        </yearOfInc>
    </primaryIssuer>

    <relatedPersonsList>
        <relatedPersonInfo>
            <relatedPersonName>
                <firstName>David</firstName>
                <lastName>Bearss, Ph.D.</lastName>
            </relatedPersonName>
            <relatedPersonAddress>
                <street1>c/o Tolero Pharmaceuticals, Inc.</street1>
                <street2>2975 Executive Parkway, Suite 320</street2>
                <city>Lehi</city>
                <stateOrCountry>UT</stateOrCountry>
                <stateOrCountryDescription>UTAH</stateOrCountryDescription>
                <zipCode>84043</zipCode>
            </relatedPersonAddress>
            <relatedPersonRelationshipList>
                <relationship>Executive Officer</relationship>
                <relationship>Director</relationship>
                <relationship>Promoter</relationship>
            </relatedPersonRelationshipList>
            <relationshipClarification></relationshipClarification>
        </relatedPersonInfo>
        <relatedPersonInfo>
            <relatedPersonName>
                <firstName>Dallin</firstName>
                <lastName>Anderson</lastName>
            </relatedPersonName>
            <relatedPersonAddress>
                <street1>c/o Tolero Pharmaceuticals, Inc.</street1>
                <street2>2975 Executive Parkway, Suite 320</street2>
                <city>Lehi</city>
                <stateOrCountry>UT</stateOrCountry>
                <stateOrCountryDescription>UTAH</stateOrCountryDescription>
                <zipCode>84043</zipCode>
            </relatedPersonAddress>
            <relatedPersonRelationshipList>
                <relationship>Executive Officer</relationship>
                <relationship>Director</relationship>
                <relationship>Promoter</relationship>
            </relatedPersonRelationshipList>
            <relationshipClarification></relationshipClarification>
        </relatedPersonInfo>
    </relatedPersonsList>

    <offeringData>
        <industryGroup>
            <industryGroupType>Pharmaceuticals</industryGroupType>
        </industryGroup>
        <issuerSize>
            <revenueRange>Decline to Disclose</revenueRange>
        </issuerSize>
        <federalExemptionsExclusions>
            <item>06b</item>
        </federalExemptionsExclusions>
        <typeOfFiling>
            <newOrAmendment>
                <isAmendment>true</isAmendment>
                <previousAccessionNumber>0001554907-14-000002</previousAccessionNumber>
            </newOrAmendment>
            <dateOfFirstSale>
                <value>2014-08-04</value>
            </dateOfFirstSale>
        </typeOfFiling>
        <durationOfOffering>
            <moreThanOneYear>false</moreThanOneYear>
        </durationOfOffering>
        <typesOfSecuritiesOffered>
            <isEquityType>true</isEquityType>
            <isOptionToAcquireType>true</isOptionToAcquireType>
        </typesOfSecuritiesOffered>
        <businessCombinationTransaction>
            <isBusinessCombinationTransaction>false</isBusinessCombinationTransaction>
            <clarificationOfResponse></clarificationOfResponse>
        </businessCombinationTransaction>
        <minimumInvestmentAccepted>0</minimumInvestmentAccepted>
        <salesCompensationList>
            <recipient>
                <recipientName>T.R. Winston &amp; Company, LLC</recipientName>
                <recipientCRDNumber>10571</recipientCRDNumber>
                <associatedBDName>T.R. Winston &amp; Company, LLC</associatedBDName>
                <associatedBDCRDNumber>10571</associatedBDCRDNumber>
                <recipientAddress>
                    <street1>1999 Avenue of the Starts</street1>
                    <street2>25th Floor</street2>
                    <city>Los Angeles</city>
                    <stateOrCountry>CA</stateOrCountry>
                    <stateOrCountryDescription>CALIFORNIA</stateOrCountryDescription>
                    <zipCode>90067</zipCode>
                </recipientAddress>
                <statesOfSolicitationList>
                    <state>CA</state>
                    <description>CALIFORNIA</description>
                    <state>CO</state>
                    <description>COLORADO</description>
                    <state>CT</state>
                    <description>CONNECTICUT</description>
                    <state>IL</state>
                    <description>ILLINOIS</description>
                    <state>MA</state>
                    <description>MASSACHUSETTS</description>
                    <state>NJ</state>
                    <description>NEW JERSEY</description>
                    <state>NY</state>
                    <description>NEW YORK</description>
                    <state>NC</state>
                    <description>NORTH CAROLINA</description>
                    <state>WI</state>
                    <description>WISCONSIN</description>
                </statesOfSolicitationList>
                <foreignSolicitation>false</foreignSolicitation>
            </recipient>
        </salesCompensationList>
        <offeringSalesAmounts>
            <totalOfferingAmount>35469236</totalOfferingAmount>
            <totalAmountSold>22490179</totalAmountSold>
            <totalRemaining>12979057</totalRemaining>
            <clarificationOfResponse>$16,702,073 new money; $5,318,870.85 conversion of convertible promissory notes (principal and accrued interest); $469,236.07 warrants to purchase common stock</clarificationOfResponse>
        </offeringSalesAmounts>
        <investors>
            <hasNonAccreditedInvestors>false</hasNonAccreditedInvestors>
            <totalNumberAlreadyInvested>48</totalNumberAlreadyInvested>
        </investors>
        <salesCommissionsFindersFees>
            <salesCommissions>
                <dollarAmount>0</dollarAmount>
            </salesCommissions>
            <findersFees>
                <dollarAmount>1485706</dollarAmount>
            </findersFees>
            <clarificationOfResponse></clarificationOfResponse>
        </salesCommissionsFindersFees>
        <useOfProceeds>
            <grossProceedsUsed>
                <dollarAmount>0</dollarAmount>
            </grossProceedsUsed>
            <clarificationOfResponse></clarificationOfResponse>
        </useOfProceeds>
        <signatureBlock>
            <authorizedRepresentative>false</authorizedRepresentative>
            <signature>
                <issuerName>Tolero Pharmaceuticals, Inc.</issuerName>
                <signatureName>/s/ Dallin Anderson</signatureName>
                <nameOfSigner>Dallin Anderson</nameOfSigner>
                <signatureTitle>President</signatureTitle>
                <signatureDate>2014-12-05</signatureDate>
            </signature>
        </signatureBlock>
    </offeringData>
</edgarSubmission>
