Bacteria have evolved distinct molecular mechanisms as a defense against oxidative stress. The foremost regulator of the oxidative stress response has been found to be OxyR. However, the molecular details of regulation upstream of OxyR remain largely unknown and need further investigation. Here, we characterize an oxidative stress and antibiotic tolerance regulator, OsaR (PA0056), produced by Pseudomonas aeruginosa. Knocking out of osaR increased bacterial tolerance to aminoglycoside and b-lactam antibiotics, as well as to hydrogen peroxide. Expression of the oxyR regulon genes oxyR, katAB, and ahpBCF was increased in the osaR mutant. However, the OsaR protein does not regulate the oxyR regulon genes through direct binding to their promoters. PA0055, osaR, PA0057, and dsbM are in the same gene cluster, and we provide evidence that expression of those genes involved in oxidant tolerance is controlled by the binding of OsaR to the intergenic region between osaR and PA0057, which contain two divergent promoters. The gene cluster is also regulated by PA0055 via an indirect effect. We further discovered that OsaR formed intramolecular disulfide bonds when exposed to oxidative stress, resulting in a change of its DNA binding affinity. Taken together, our results indicate that OsaR is inactivated by oxidative stress and plays a role in the tolerance of P. aeruginosa to aminoglycoside and b-lactam antibiotics.