A Pseudomonas aeruginosa mutant strain M122 was isolated from a Mu transposon insertion mutant library. In our prophase research, we have found that PA0058, a novel gene encodes a 234-residue conserved protein, was disrupted in the M122 mutant. In this study, the bacteriostatic experiment in vitro indicates that M122 has abnormally high aminoglycoside resistance. We expressed PA0058 in E. coli and found that PA0058 oxidizes and reduces disulfide. This biochemical characterization suggests that PA0058 is a novel disulfide oxidoreductase. Hence, the protein was designated as DsbM. Microarray analysis of the M122 mutant showed its unusual phenotype might be related to the bacterial antioxidant defense system mediated by the oxyR regulon. Meanwhile, we detected -SH content in the periplasm of M122 and wild strain and found a lower -SH/S-S ratio in M122. Therefore, we consider that the loss of dsbM function decreased the -SH/S-S ratio, which then prolongs the OxyR-regulated response, thereby conferring high aminoglycoside resistance to the M122 mutant strain. Our findings have important implications for understanding the mechanisms underlying aminoglycoside resistance in P. aeruginosa.