Pseudomonas aeruginosa is a gram negative bacterium, which can cause nosocomial infection, particularly in immunocompromised patient. Regulation of gene expression at post-transcriptional level has been shown to be crucial for oxidative stress adaptation. P. aeruginosa TrmA shows about 50% identity to m5U methyltransferase enzyme or TrmA in E. coli. Recombinant P. aeruginosa TrmA methylated tRNAs extracted from P. aeruginosa using in vitro methyltransferase assay, in which SAM was used as a methyl donor. Using disk diffusion and killing assay, trmA mutant was more sensitive to hydrogen peroxide (H2O2) than P. aeruginosa wild-type. The H2O2 sensitive phenotype could be complemented by introducing the trmA full length gene into the trmA mutant strain. Catalase activity was determined, since this is a main enzyme detoxifying H2O2. Interestingly, the trmA mutant had lower catalase activity than that of the wild-type and trmA complemented strains, supporting the H2O2 sensitive phenotype of the the trmA mutant. The increased TrmA protein expression concurs with the increased m5U modification, when exposed to H2O2, demonstrating a possible function of TrmA-catalyzed m5U formation in response to oxidative stress. Altogether, TrmA possesses methyltransferase activity for the formation of m5U, and the TrmA-mediated m5U may play a role in oxidative stress response.