Factor Xa (fXa) is the vitamin K-dependent serine protease of the prothrombinase complex (fXa, factor Va, negatively charged membrane, and calcium) which is responsible for the conversion of prothrombin to thrombin in the final stage of the coagulation cascade. The proteolytic activity of fXa in plasma is primarily regulated by three physiological inhibitors, antithrombin (AT), protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI). The first two inhibitors belong to the serpin family of plasma inhibitors, both of which require cofactors for their effective interaction with fXa. Thus, the AT interaction with the heparin-like glycosaminoglycans on the surface of the endothelium, and the ZPI complex formation with protein Z on membrane phospholipids is required for the physiological regulation of fXa by both serpins. On the other hand, TFPI is a slow and tight-binding, Kunitz type inhibitor that is capable of rapidly inhibiting fXa independent of a cofactor. This article will review the structural features that enable fXa to specifically interact with these three inhibitors under different conditions.