Antimicrobial peptides have been known to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against invading pathogenic microbes. Parasin I, a potent 19-residue inducible antimicrobial peptide, was recently isolated from the skin mucus of wounded catfish, Parasilurus asotus. The amino acid sequence of parasin I was identical to the N-terminal region of histone H2A. Previous study suggested that parasin I might be produced from histone H2A by an unknown proteolytic cleavage. In this study, we demonstrated the mechanism of parasin I production in catfish skin mucosa upon epidermal injury and assessed the biological role of parasin I in the innate host defense. Cathepsin D was found to exist as an inactive proenzyme (procathepsin D) in the mucus, and a metalloprotease, induced upon injury, activated the procathepsin D to active cathepsin D, which consequently cleaved the $Ser^19-Arg^20$ bond of histone H2A to produce parasin I. Using recombinant procathepsin D as a substrate, we identified the procathepsin D-processing metalloprotease in the mucus of wounded catfish. The purified enzyme, named Pa-metalloprotease, carried out the activation of procathepsin D to mature cathepsin D by cleaving specifically the $Phe^290-Gly^291 bond of procathepsin D. Sequence analysis of the cDNA encoding Pa-metalloprotease showed that Pa-metalloprotease had a significant homology with matrix metalloproteinases. Immunohistochemical analysis revealed that unacetylated histone H2A, a precursor of parasin I, and procathepsin D were present in the cytoplasm of epithelial mucous cells, and parasin I was produced and secreted to the mucosal surface upon epidermal injury. Western blot analysis showed that parasin I was also present in the skin mucus of other fish species. Furthermore, parasin I showed a strong antimicrobial activity against fish-specific pathogens. Together, these results indicate that Pa-metalloprotease and cathepsin D are invol...