[Objective] We studied the hemolytic activity of Vibrio alginolyticus strains, and the distribution of a hemolysin gene vah in these strains; we analyzed the functions of vah gene and the related promoter, and assessed their contributions to the hemolytic activity. [Methods] Hemolytic test was performed using 47 V. alginolyticus strains, including one type strain 1.1587 and 46 environmental strains isolated from seawater and various marine animals. The difference of hemolytic ability was compared among a wild V. alginolyticus strain ZJ051 that showed hemolytic activity, a recombinant Escherichia coli BL21 aiming at a predicted vah gene, a vah-deletion mutant, and a vah-recovery strain. The distribution of vah gene was further detected in V. alginolyticus followed by the sequence analysis of vah genes and their promoters among the hemolytic and non-hemolytic strains. [Results] Among the V. alginolyticus strains 47.8% showed hemolytic activities, and thus hemolysis was ubiquitous in environmental V. alginolyticus strains. Hemolysis was observed in the recombinant Escherichia coli BL21 and the vah-recovery strain but not in the vah-deletion mutant. The vah gene widely distributed in V. alginolyticus. The vah sequences of V. alginolyticus strains were highly similar, and they shared identical amino acid sequences. Through the vah promoter comparison between the hemolytic and non-hemolytic strains, a difference in 188-190 base sites of the promoters was discovered. [Conclusion] Hemolysis is directly caused by the vah gene, and the discrepancy of hemolytic ability among various V. alginolyticus strains has no relation with the difference of vah sequences, which is likely determined by the diversity in 188-190 base sites of vah promoters.