The halophilic marine bacterium, Vibrio vulnificus, occasionally causes fatal septicemia in immunocompromised patients. Mice are commonly used as experimental animals to investigate the virulence of V. vulnificus, however, a large number of mice are generally required for bioassays. The present study examined whether the invertebrate species, silkworms, can be used instead of mice to investigate V. vulnificus virulence. When the silkworms were inoculated with 1.2x107 colony forming units of V. vulnificus OPU1 Rf, a virulent strain of V. vulnificus, all injected silkworms died within 48 h, however, those injected with culture filtrate or diluent did not. This silkworm infection model was then used to isolate attenuated V. vulnificus mutants from 1,016 transposon inserted mutants. Consequently, a harmless mutant, SW998, was isolated. In this strain, the transposon was inserted into the rtxA gene, which is a known V. vulnificus virulence gene. In conclusion, the present study demonstrated that silkworms are useful animals for investigating the virulence of V. vulnificus.