The virulence gene vah1 in Vibrio anguillarum was knocked out by gene recombination, and the strain Δvah1 with vah1 deletion was constructed in this study. The hemolytic activity of Δvah1 strain decreased by 89.4% and the biofilm forming ability decreased by 16%. The median lethal dose (LD₅₀) of Δvah1 strain infected Litopenaeus vannamei was (9.77±0.50)×10⁵ cfu/mL, compared with (3.16±0.80)×10⁵ cfu/mL of the LD₅₀ of the wild strain of V. anguillarum,it increased by 2.09 times; the colonization ability of Δvah1 strain in L. vannamei decreased evidently after vah1 deletion. Compared with the wild strain of V. anguillarum, the destruction degree of Δvah1 strain on hepatopancreas cells was significantly smaller. After Δvah1 strain infected L. vannamei, the expression levels of immune related genes CAT, SOD, PO, LZM and ACP in hepatopancreas decreased to some extent. These results showed that the hemolysis and pathogenicity to L. vannamei of the vah1 deficient V. anguillarum strain were reduced in varying degrees, which laid a foundation for the preparation of subsequent attenuated vaccines or specific immune enhancers against V. anguillarum.