Protein-protein interactions (PPIs) are key mechanisms in the maintenance of biological regulatory networks. Herein, we characterize PPIs within ToxR and its co-activator, ToxS, to understand the mechanisms of ToxR transcription factor activation. ToxR is a key transcription activator that is supported by ToxS for virulence gene regulation in Vibrio cholerae. ToxR comprises a cytoplasmic DNA-binding domain that is linked by a transmembrane domain to a periplasmic signal receiver domain containing two cysteine residues. ToxR-ToxR and ToxR-ToxS PPIs were detected using an adenylate-cyclase-based bacterial two-hybrid system approach in Escherichia coli. We found that the ToxR-ToxR PPIs are significantly increased in response to ToxR operators, the co-activator ToxS and bile salts. We suggest that ToxS and bile salts promote the interaction between ToxR molecules that ultimately results in dimerization. Upon binding of operators, ToxR-ToxR PPIs are found at the highest frequency. Moreover, disulfide-bond-dependent interaction in the periplasm results in homodimer formation that is promoted by DNA binding. The formation of these homodimers and the associated transcriptional activity of ToxR were strongly dependent on the oxidoreductases DsbA/DsbC. These findings show that protein and non-protein partners, that either transiently or stably interact with ToxR, fine-tune ToxR PPIs, and its associated transcriptional activity in changing environments.