Antibiotic-resistant Pseudomonas aeruginosa(PAO1) poses a significant challenge in clinical infections, highlighting the importance of studying its resistance mechanisms for improving clinical treatments. Our study found that the ΔprtN exhibited significant resistance to trimethoprim (Tmp) compared to the wild type. To elucidate the underlying mechanism of this resistance, we assessed the expression of folA, the target of Tmp. Interestingly, folA expression in ΔprtN was not elevated, instead but rather decreased compared to the PAO1 strain. Further investigations revealed that a double mutant lacking both the PrtN-regulated S type pyocin biosynthesis gene and prtN exhibited slightly reduced Tmp resistance. In contrast, the lipopolysaccharide-deficient strain ΔwbpL showed slightly increased Tmp resistance.In ΔprtN, the expression levels of reactive oxygen species(ROS)-related genes (oxyR, katA, ahpC), biofilm formation, and antibiotic resistance associated with efflux pumps showed no significant difference compared to the wild type.In conclusion, the Tmp resistance observed in the prtN mutant is likely due to the regulatory effects of PrtN on S-type pyocins and lipopolysaccharide-related targets.