Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fII(WE)), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)-dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fII(WE) mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fII(WE/WE) mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fII(WT/WE) mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl₃ vessel injury. More interestingly, prothrombin(WE) expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombin(WE) also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.