Vibrio alginolyticus,a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host,but few attenuated vaccine against V. alginolyticus is available. The type III secretion system( T3SS),an important pathogenic factor of V. alginolyticus,is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3 SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface. vscO locates at the “ needle” site of T3 SS,playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper,an in-frame deletion mutant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol( Cm) and sucrose screening. The LD50 changes of ZJ03ΔvscO mutant strains compared with the strain ZJ03 were examined in grouper( Epinephelus coioides). The ZJ03ΔvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJ03ΔvscO in E. coioides through injection and immersion,the specific antibody titers were markedly higher than that in the saline control group( P < 0. 05). The titers of injection and immersion group on the forth week reached the maximums at 1∶ 2 048 and 1∶ 128,respectively. The relative percentage survival( RPS) of injection group was 84%,while that in immersion group was 68%.These results indicate that the ZJ03ΔvscO of V. alginolyticus has a high immunogenicity,and can be used as live attenuated vaccine. In addition,RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus. Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach prevented V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system (T3SS), an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3 SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface. vscO locates at the “needle ” site of T3 SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mutant of vscO was constru The LD50 changes of ZJ03ΔvscO mutant compared with the strain ZJ03 were examined in grouper (Epinephelus coioides). The ZJ03ΔvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJ03ΔvscO in E. coioides through injection and immersion, the specific antibody titers were markedly higher than that in the saline control group (P <0.05). The titers of injection and immersion group on the forth week reached the maximums at 1: 2 048 and 1: 128, respectively. The relative percentage survival (RPS) of injection group was 84% ​​while while that in immersion group was 68%. These results results that the ZJ03ΔvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide techn ical support for controlling fish diseases caused by V. alginolyticus.