We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human αIIbβ3. RUC-1 did not inhibit αVβ3, suggesting that it interacts with αIIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the αIIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid αIIbβ3 receptor composed of human αIIb and murine β3, but not a hybrid receptor composed of murine αIIb and human β3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid hαIIb/mβ3 receptors. Collectively, these data support RUC-1's specificity for αIIb, provide new insights into the αIIb binding pocket, and establish RUC-1's antithrombotic effects in vivo.