Granzymes are key components of the cytotoxic arm of the immune response, which play critical roles in eliminating host cells infected by intracellular pathogens and transformed cells. Although the induction of cell death is likely a central process underlying the function of these enzymes, little is known about whether granzymes use additional mechanisms to exert their antipathogen activity. This study identifies La, a phosphoprotein involved in multiple roles in cellular and viral RNA metabolism, as the first nonapoptotic substrate of granzyme H (gzmH), a cytotoxic granule protease that is constitutively expressed by NK cells. Cleavage of La by gzmH occurs at Phe-364 (P(1) site) and generates a COOH-terminal truncated form of La that loses nuclear localization and decreases HCV (hepatitis C virus)-internal ribosome entry site (IRES)-mediated translational activity. The ability of gzmH to cleave host proteins involved in essential viral functions provides a novel mechanism by which granzymes can mediate direct antiviral activities.