[Objective] To investigate the role of kdpD gene in Vibrio alginolyticus, a kdpD gene deletion mutant was generated. [Methods] Combining with chloramphenicol (Cm) and sucrose screening, we constructed an in-frame deletion mutant of kdpD using overlap PCR and homologous recombination technology. We then investigated the effect of the mutant on its growth, ECPase activity and virulence in fish. [Results] The mutant ΔkdpD was successfully constructed. The mutant ΔkdpD showed no differences of ECPase activity and growth from the wild-type strain. But a decrease in swarming ability and biofilm formation was found. Moreover, virulence assays with zebrafish model confirmed that virulence of the mutant declined by 8.84 times. [Conclusion] The kdpD gene could regulate swarming ability, biofilm formation and virulence, but it has no effect on growth and ECPase activity.