<p>Echinatin (Ecn), a potent bioactive compound extracted from the roots and rhizomes of licorice, demonstrates anti-oxidant, anti-cancer, cardioprotective, anti-neurotoxic, and hepatoprotective properties. This study aims to investigate the antithrombotic potential of Ecn and clarify its mechanisms of action. Its anticoagulant effects were assessed via activated partial thromboplastin time, prothrombin time, thrombin and factor Xa (FXa) activities, as well as fibrin formation and platelet aggregation. Additionally, the effect of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator was evaluated in tumor necrosis factor (TNF)-α–stimulated human endothelial cells. Our experiments revealed that Ecn exhibited antithrombotic activity comparable to that of rivaroxaban, a well-established direct FXa inhibitor, particularly in suppressing FXa activity and platelet aggregation induced by adenosine diphosphate (ADP) and U46619, a synthetic thromboxane A2 analog. Ecn also reduced the surface expression of P-selectin, inhibited phosphorylation of myristoylated alanine-rich C kinase substrate, and decreased PAC-1 activation following ADP or U46619 stimulation. Furthermore, Ecn enhanced nitric oxide production while preventing excess endothelin-1 release in HUVECs exposed to these agonists. In vivo experiments in mouse models of arterial and pulmonary thrombosis demonstrated that Ecn is a potent anticoagulant and antithrombotic agent. These findings indicate that Ecn could serve as a promising lead compound for developing innovative anti-FXa and antiplatelet therapeutics.</p>