Abstract: Objective: Exploring the anti-thrombotic effect and mechanism of nattokinase(NK) in vitro and vivo. Methods: The thrombolytic effect of NK on old thrombus was studied by blood clot lysis method in vitro, the rat carotid artery thrombosis model induced by ferric chloride and the rat venous thrombosis model induced by inferior vena cava ligation method were used to study the thrombolytic effect of NK in vivo. Thromboxane(TXB₂), 6-keto-prostaglandin F1α(6-K-PGF1α), prostaglandin E₂(PGE₂), tissue type plasminogenactivator(t-PA), \cdot \rmO_2^- , ·OH, xanthine oxidase(XOD) and other indicators were detected to explore the antithrombotic mechanism of NK. Results: Nattokinase could significantly dissolve old thrombus (P<0.05) in vitro, and had a strong thrombolytic effect in vivo, and its thrombolytic effect was stronger than that of urokinase. Nattokinase could significantly affect the values of TXB₂, 6-K-PGF1α, PGE₂, t-PA, \cdot \rmO_2^- , ·OH and XOD. The values of TXB₂, PEG₂, \cdot \rmO_2^- , ·OH and XOD increased significantly(P<0.05) , while the values of 6-K-PGF1α and t-PA decreased significantly(P<0.05) . Conclusion : NK had strong thrombolytic activity in vivo and vitro, and had effect on fresh thrombus and old thrombus. NK could inhibit thrombosis by inhibiting platelet aggregation, reducing oxidative damage and reducing the level of coagulation factors. NK could also play a direct thrombolytic role by stimulating the production of t-PA, dissolving fibrin, and might play an indirect thrombolytic role by inhibiting the production of plasminogen activator inhibitor 1(PAI-1).