Stenotrophomonas maltophilia is a cause of nosocomial infections especially in the immunocompromised patients. It inherits several antibiotic resistance mechanisms. Understanding the antibiotic resistance mechanisms is crucial for development of alternative treatments. Transcriptional factor SoxR is known as superoxide sensing regulator, which controls several oxidative stress response genes, including superoxide dismutase gene. Our finding revealed that SoxR in S. maltophilia regulates not only sod genes, but also the major facilitator superfamily, mfsA, gene. MfsA functions as an efflux pump, which is responsible for resistance to several compounds. When S. maltophilia contains plasmid-mediated overexpression of mfsA, the bacteria showed an increased resistance not only to paraquat but also to fluoroquinolone antibiotics. Moreover, heterologous expression of mfsA in other Gram-negative pathogens conferred resistance to paraquat as well as to fluoroquinolones. In S. maltophilia, oxidized SoxR, a consequence of exposure to superoxide stress, could increase mfsA expression, leading to multidrug resistance. We hypothesized that mutation of SoxR to the form that is easily oxidized would up-regulate genes contributing to both oxidative stress protection and multiple antibiotic resistance, thereby enhancing bacterial virulence and its ability to survive in hostile conditions. By using random mutagenesis methods, we got 5 groups of SoxR mutations that lead to antibiotic resistances. Some of these mutations were selected and confirmed by site-directed mutagenesis. The structural analysis of the mutated SoxR is required for better understanding of the mechanism at molecular level.