Thrombosis and thromboembolic occlusions of major and minor blood vessels are a major complication in various peripheral vascular diseases. Antiplatelet agents, key tools in the treatment of atherothrombosis, therefore became a mainstay medication for a wide range of vascular diseases. Hyperoside, an active compound from the Rhododendron brachycarpum G. Don (Ericaceae), was reported to have antioxidant, anti-hyperglycemic, anticancer, anti-inflammatory, and anti-coagulant activities. However, antiplatelet properties of hyperoside have not been studied. In this study, the antiplatelet activities of hyperoside were measured by thrombin- or collagen-induced platelet aggregation in vitro, adenosine diphosphate-induced platelet aggregation in vivo, and the thrombus formation in vivo. Our data showed that in vitro assays using freshly isolated human platelets, hyperoside showed the inhibition of the thrombin- or collagen-induced platelet aggregation. In accordance with these enhanced in vitro antiplatelet activities, hyperoside showed enhanced anti-thrombotic effects in vivo pulmonary embolism model and arterial thrombosis model. Collectively, these results indicate that hyperoside possesses antiplatelet activities and might offer enhanced anti-thrombotic efficacies without increasing side effects.