For better understanding of the host infection mechanism of Vibrio, a Vibrio parahaemolyticus collagenase mutant was generated by insertional inactivation of a vppC gene encoding extracellular collagenase. A recombinant DNA containing vppC::nptII was cloned into a suicide plasmid pDMS197, resulted in pVCM03. The recombinant suicide plasmid pVCM03 contained in E. coli $chi$7213 was transferred to a wild-type V. parahaemolyticus 04 through conjugation. The recombinant vppC::nptII DNA in pVCM03 was exchanged with wild-type allele by homologous recombination resulting vppC mutant, V. parahaemolyticus CM. The mutant was selected and screened on TCBS media containing 10% sucrose and kanamycin. The mutation by allele exchange was confirmed with the comparison of the size of DNAs amplified by PCR. V. parahaemolyticus CM showed at least 4-fold less collagen-degrading activity than those of wild-type, and the mutant exhibited less cytotoxicity than that of wild-type in MTT assay.