Thrombin is an essential serine protease for survival. Since the discovery of heparin in the early twentieth century, significant advances have been made in the understanding of thrombin structure and function in coagulation system. Endogenous anticoagulant proteins in blood tightly regulate thrombin generation, but additional anticoagulant agents may be necessary to suppress excessive thrombin formation or defective anticoagulant proteins. Despite the availability of an array of anticoagulant agents based on chemical and biological engineering technologies, anticoagulation therapy remains a challenge for clinicians in terms of balancing bleeding and thrombosis. The aim of this article is to review endogenous serine protease inhibitors and novel antithrombotic agents in relation to pharmacologic regulation of thrombin.