Vibrio alginolyticus, a Gram-negative bacterium has been described to be the most common and important pathogen isolated from diseased fish and shellfish.To identify antigens associated with virulence, we compared total soluble proteins extracted from a virulent(ZJ03)and an avirulent (ZJ12) of V.alginolyticus.Total soluble proteins extracted were separated by two dimensional differential gel electrophoresis (2D-DIGE).With the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), 68 protein spots which were unique to strain ZJ03 were identified.The largest proportion of these proteins was metabolism-related.2 of the proteins in the virulent strain ZJ03 are known virulence-associated factors: Omp W and Omp TolC.26 of the identified proteins are putative virulence-associated factors or vaccine candidates.These findings contribute to the understanding of V.alginolyticus pathogenic mechanisms.