The cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt), Ctsb(-/-), Ctsl(-/-), and Ctss(-/-) mice with the virulent reovirus strain T3SA+. The survival rate of Ctsb(-/-) mice was enhanced in comparison to that of wt mice, whereas the survival rates of Ctsl(-/-) and Ctss(-/-) mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed in Ctsl(-/-) and Ctss(-/-) mice in comparison to the levels for wt and Ctsb(-/-) mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.