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Hyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. In this study, human medical examination showed that elevated human plasma Hcy levels correlated positively with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation in apolipoprotein E-deficient (ApoE−/−) mice with acute or chronic HHcy. Upon homocysteine (Hcy) administration in C57BL/6J mice, platelet aggregation, spreading and clot retraction were markedly induced. More important, Hcy increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE−/− mice. In this study, we identified a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently exacerbating thrombosis and the results revealed an innovative approach to treating HHcy-related thrombotic diseases.