Abstract: This study aimed to investigate the effects of PmrB_△Leu172 on polymyxin B resistance in Pseudomonas aeruginosa. Pseudomonas aeruginosa PmrB_△Leu172 strain was constructed by homologous recombination technology, and the minimum inhibitory concentration of polymyxin B against wild type and mutant strains were determined by the broth microdilution technique. The effect of PmrB_△Leu172 mutation on gene transcription level was determined by RNA sequencing. Reverse transcription quantitative PCR was used to verify the effect of PmrB_△Leu172 mutation on the transcription level of PmrB-regulated genes in P. aeruginosa. The results showed that the minimum inhibitory concentration and minimum bactericidal concentration of mutant strain were 4 and 2 times higher than that of wild-type, respectively, and the transcription level of PmrB-regulated genes was also up-regulated. The results suggested that PmrB_△Leu172 mutation of P. aeruginosa can enhance the regulation of PmrA-PmrB two-componentsystem, which leads to the resistance of P. aeruginosa to polymyxin B.