Using endoplasmic reticulum oxidoreductase 1α (ERO1α) conditional knockout (CKO) mice, a recent study underscores the crucial role of ERO1α in platelet activation under thrombotic conditions. Through a high-throughput screen of 39,901 compounds, we identify M6766 as a selective inhibitor of ERO1α with an IC50 of 1.4 μM and a KD of 1.1 μM. A docking model and biochemical studies reveal that M6766 binds to the flavin adenine dinucleotide-binding pocket in ERO1α and exhibits >70-fold selectivity over other tested enzymes, except ERO1β, which it inhibits with an IC50 of 7.2 μM. M6766 concentration-dependently inhibits granule secretion, αIIbβ3 integrin activation, Ca2+ mobilization, and platelet aggregation induced by various agonists, but it does not affect agonist-induced production of reactive oxygen species. Pretreatment of ERO1α with M6766 reduces its binding to the Ca2+ sensor stromal interaction molecule 1. To validate whether these inhibitory effects result from the inhibition of ERO1α and ERO1β, we generate megakaryocyte-specific Ero1β or Ero1α/β CKO mice. Deletion of platelet Ero1α/β impairs platelet activation and aggregation, whereas deletion of Ero1β has no effect. While EN460 markedly inhibits the function of Ero1α/β-null platelets, M6766 does not, highlighting its specificity. M6766 treatment diminishes platelet accumulation on collagen-coated surfaces under arterial shear conditions. Moreover, intravenous injection of M6766 into mice decreases arterial thrombosis and infarct volume during ischemic stroke without prolonging tail bleeding times. Although eptifibatide, an αIIbβ3 antagonist, effectively blocks arterial thrombosis, it prolongs bleeding times at therapeutic doses. Our findings suggest that ERO1α inhibition is a promising anti-thrombotic strategy with potential advantages over current therapies.