Motility is thought to contribute to the virulence of many pathogenic bacteria. Infection studies in many motile pathogens found that in many causes normal motility was required for efficient infection. However, there are few reports on the direct correlation between motility and virulence in Vibrio. In order to understand the role of motility of Vibrio alginolyticus in virulence, the mini-Tn10/Km transposon-induced mutant bank was constructed and 73 motility defective mutants were screened out by double semi-soft agar medium. After purification and repeated screening the mutant ND-01MM with stable motility defect was obtained and identified by Southern blot. The chemotaxis and adhesion of wild-type strain ND-01 and the mutant ND-01MM to mucus of the natural host Pseudosciaena crocea and their persistence in phagocytes of P. crocea were tested. The results showed that the chemotaxis and adhesion of the mutant was highly significantly impaired(P<0.01)when compared with the wild-type strain, however there was no significant difference in their ability of persistence in phagocytes(P>0.05). The results of infection test showed that the virulence of the mutant was decreased by immersion infection, but not different from the wild-type strain by intraperitoneal injection.