Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of arctigenin in human platelets and attempted to identify the mechanism responsible for thromboxane A₂ (TXA₂) generation, integrin α_IIbβ₃ activation and cAMP pathway. Arctigenin exhibited obvious inhibitory effects on collagen-, thrombin-, and ADP-induced human platelet aggregation, granule secretion, TXA₂ generation, integrin α_IIbβ₃ activation, and clot retraction. Additionally, we found that arctigenin attenuated PI3K/Akt/mTOR/GSK-3β and MAPK signaling pathways, and increased cAMP level. Accordingly, the findings support that arctigenin attenuates thrombotic events through the inhibition of platelet activation and platelet plug formation. Therefore, we suggest that arctigenin may have therapeutic potential as an antiplatelet and antithrombotic agent.