The fundamental importance of thrombin in biology and medicine has made it one of the most extensively studied of all proteases. Thrombin performs essential functions in vertebrate biology as the central enzyme involved in blood coagulation and platelet aggregation, and as a mitogen and secretagogue for a variety of cell types. Thrombin is synthesized in the liver and secreted into the general circulation in an inactive zymogen form (prothrombin), a complex multidomain glycoprotein that is activated to yield thrombin at sites of vascular injury by limited proteolysis following upstream activation of the coagulation cascade. Thrombin shares its general architecture and catalytic mechanism with those of pancreatic trypsin, the prototypical digestive serine protease. However, the specificity of thrombin toward substrates and cofactors, as well as its spatiotemporal regulation by effectors and inhibitors, is directed by features of the molecule that distinguish it from relatively nonspecific serine proteases like trypsin. Structural and functional studies have demonstrated the presence of surface loops that partially occlude the active site and make specific contacts with residues adjacent to the scissile bond of substrates. Specificity toward macromolecular substrates and cofactors is additionally enhanced by anion-binding exosites that are spatially distinct from the active site. More than five decades of multidisciplinary research on thrombin have produced an abundance of functional and structural information and provided a robust framework for understanding the role of thrombin in vertebrate biology.