Enterococcus faecalis was isolated from the body fluid of dead Galleria mellonella larvae. Upon injection of E. faecalis into the hemocoel of G. mellonella, the bacteria destroyed parts of humoral defense systems in the hemolymph. In a test for the proteolytic activity of E. faecalis CS, it was confirmed that the enzyme degraded three well-known a-helical antimicrobial peptides, cecropin A, melittin and halocidin, and abolished their activities. We also determined putative cleavage sites on the primary sequences of three peptides through purification and mass analysis of peptide fragments digested by E. faecalis CS. Furthermore it was found that apolipophorin-III, recently known as a critical recognition protein for invading microbes in the hemolymph of G. mellonella, was also degraded by E. faecalis CS. Taken together, the present work shows that the protease in secretions from E. faecalis destroyed two critical humoral immune factors in the hemolymph of G. mellonella larvae. In addition, this paper demonstrates that the relationship between the host insect and the pathogenic bacteria might provide a valuable model system to study the enterococcal virulence mechanism, which may be relevant to mammalian pathogenesis.