The relationship between extracellular chymoelastase (Pr1) and the virulence of Metarhizium anisopliae for Manduca sexta was studied using either IgG antibodies vs Pr1 or turkey egg white inhibitor, a potent specific inhibitor of Pr1. Treatment of M. sexta with the inhibitor during infection significantly delayed mortality. The inhibitor also reduced melanization of cuticle (an early host response to infection) and invasion of the hemolymph, as well as maintained the host's growth rate. The incorporation of antibodies or inhibitor delayed penetration of cuticle, but did not affect spore viability or prevent growth and formation of appressoria on the cuticle surface. This suggests that inhibition of Pr1 reduced infection by limiting fungal penetration of the insect cuticle. In vitro studies using inhibitor showed that the accumulation of protein degradation products from cuticle, including ammonia, was dependent on active Pr1. This confirms the major role of Pr1 in solubilizing cuticle proteins and making them available for nutrition. It is concluded that Pr1 serves as a virulence factor by causing the localized destruction of cuticle proteins, which enables rapid invasion of the host with concomitant provision of nutrients.