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Vibrio anguillarum, a Gram-negative bacterial pathogen, is the causative agent of vibriosis that affects a wide range of aquatic animals. In this study, we obtained a mutant V. anguillarum, C312M, derived from the pathogenic V. anguillarum C312 by selection of rifampicin resistance. C312M was slower in growth than the wild type C312, particularly under conditions of iron depletion. Compared to C312, C312M was altered in protein production profile and exhibited a dramatically increased median lethal dose. Safety analysis showed that C312M was stable in virulence in the absence of selective pressure. To examine the potential of C312M as a live attenuated vaccine, Japanese flounder Paralichthys olivaceus were vaccinated with C312M via oral, immersion, and oral plus immersion routes. Microbiological analysis showed that C312M was recovered from the gut, liver, kidney, and spleen of the vaccinated fish in 1 to 14 d post-vaccination. When the fish were challenged with C312 at 1 mo post-vaccination, C312M-vaccinated fish exhibited relative percent survival rates of 60 to 84%. Comparable protection was observed when the fish were challenged with a heterologous V. anguillarum strain. Further analysis showed that C312M-vaccinated fish produced specific serum antibodies which enhanced serum bactericidal activity in a manner that is probably complement-dependent. These results indicate that C312M is highly attenuated in virulence but still retains residual infectivity, and that C312M is an effective vaccine when delivered alive via immersion and oral feeding.