[Objective] In this study, we demonstrated the mechanism of ToxR activating virulence gene expression by modifying its protein function in Vibrio cholerae. [Methods] ToxR redox status assay with or without DsbA was analyzed by thiol-trapping assay. ToxR cysteine mutant ToxR_C263/293S was obtained by site-directed mutagenesis. Escherichia coli strains containing ctxAB promoter luxCDABE transcriptional fusion and a plasmid-coded ToxR expressed under the control of arabinose were used to analyze the transcriptional level. ToxR and ToxS proteins interaction was detected by the bacterial two-hybrid system. [Results] The two cysteines in the periplasmic domain of ToxR could be oxidized by DsbA. When ToxR and ToxS were co-transcribed under the control of the same promoter, ToxR activated ctxAB expression to a higher level in E. coli dsbA knock out mutant. However, when ToxR was expressed alone without ToxS, the redox status of ToxR had no effect on its activation of ctxAB expression in E. coli. Bacterial two-hybrid system assay showed that ToxS greatly enhanced ToxR homodimerization regardless of the redox status of ToxR, and DsbA strongly repressed ToxS-ToxS interaction. [Conclusion] The redox status of ToxR has no effect on its activating virulence gene expression and ToxS enhances ToxR activity by promoting ToxR homodimerization. DsbA indirectly affects ToxR activating virulence genes expression by repressing ToxS homodimerization.