The plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA(-/-)) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA(-/-) animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1(-/-) mice compared with wild-type mice and the PAI-1(-/-) animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA(-/-) mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.