Abstract: Vibrio harveyi is an important pathogen of aquatic animals. In order to study the biological characteristics of the strain after the absence of hemolysin gene vhh, the knock-out mutant of vhh in V. harveyi was constructed by homologous recombination technique. Moreover, the biological characteristics of the wild type strain and the mutant strain were compared. The results show that the deletion of vhh gene did not affect the growth of the strain, neither the secretion of extracellular protease, the stress response to H₂O₂ and Cu²⁺, the uptake and utilization of iron, and the 15 antibiotics resistance and biofilm formation, but it caused the strain to swim and surge significantly. In addition, although vhh was highly expressed in the wild type strain, the strain did not show hemolytic activity. The results indicate that vhh negatively regulates the bacterial motility. The study provides novel information for understanding the function of vhh gene in V. harveyi.