ABSTRACTPolymyxins are currently the last-resort antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections. To expand the understanding of the intrinsic resistance mechanism against polymyxins, a laboratory strain ofPseudomonas aeruginosaPAO1 was subjected to serial passage in the presence of sublethal doses of polymyxin B over a period of 30 days. By whole-genome sequencing of successively isolated polymyxin B-resistant isolates, we identified a frameshift mutation (L183fs) in themvfRgene that further increased polymyxin resistance in thepmrBmutant background. A ΔmvfRmutation alone showed higher tolerance to polymyxin B due to altered lipopolysaccharide (LPS) on the surface of bacterial cells, which decreases its outer membrane permeability. In the ΔmvfRmutant, polymyxin B treatment caused the upregulation ofrfaD, the gene involved in LPS core oligosaccharide synthesis, which is responsible for polymyxin tolerance. To the best of our knowledge, this is the first report ofmvfRmutation conferring polymyxin resistance inP. aeruginosavia increased integrity of bacterial outer membrane.IMPORTANCEAntibiotic resistance imposes a considerable challenge for the treatment ofP. aeruginosainfections. Polymyxins are the last-resort antibiotics for the treatment of multidrug-resistantP. aeruginosainfections. Understanding the development and mechanisms of bacterial resistance to polymyxins may provide clues for the development of new or improved therapeutic strategies effective againstP. aeruginosa. In this study, using anin vitroevolution assay in combination with whole-genome sequencing, we demonstrated that MvfR controls tolerance to polymyxin B by regulating therfaDgene inP. aeruginosa. Our results reveal a novel mechanism employed byP. aeruginosain the defense against polymyxin antibiotics.