Vibrio parahaemolyticus transcriptional regulator vtrB gene knockout and complementary strains were constructed, and the role for VtrB in bile resistance was also explored. Two pairs' primers were designed according to the vtrB gene in GenBank. The upstream and downstream flanking DNA fragments of vtrB gene were fused by PCR, and then cloned into the suicide plasmid pDS132. The recombinant suicide vector was transferred from E. coli S17-1 λpir to V. parahaemolyticus for homologous recombination. The vtrB gene mutants were screened by 10% sucrose. Complement strain C-ΔvtrB was constructed by transferring pBAD33-vtrB into ΔvtrB mutant strain. The survival rates of wild type, ΔvtrB mutant and C-ΔvtrB strains cultured in media containing 2% deoxycholate (DOC) were determined. Based on the principle of homologous recombination, the PCR amplification and transcription level detection results of the vtrB gene showed that V. parahaemolyticus vtrB gene mutant ΔvtrB and its complementary strain C-ΔvtrB were successfully constructed. The survival rates of the ΔvtrB strain was 17%, 4% and 1% in the presence of 2% DOC for 20 min, 40 min and 60 min, respectively. This was a significantly lower level of survival than that of the wild type and C-ΔvtrB strains (P<0.001). The results indicated that the VtrB is helpful to the bile resistance of V. parahaemolyticus.