Using comparative genome sequencing analysis, we identified a novel mutation in Bacillus subtilis that confers a low level of resistance to fusidic acid. This mutation was located in the mdtR (formerly yusO) gene, which encodes a MarR-type transcriptional regulator, and conferred a low level of resistance to several antibiotics, including novobiocin, streptomycin, and actinomycin D. Transformation experiments showed that this mdtR mutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream gene mdtP (formerly yusP), which encodes a multidrug efflux transporter, is cotranscribed with mdtR as an operon. Disruption of the mdtP gene completely abolished the multidrug resistance phenotype observed in the mdtR mutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to the mdtRP promoter, thus leading to repression of its transcription. Moreover, gel mobility shift analysis indicated that an Arg83 --> Lys or Ala67 --> Thr substitution in MdtR significantly reduces binding affinity to DNA, resulting in derepression of mdtRP transcription. Low concentrations of fusidic acid induced the expression of mdtP, although the level of mdtP expression was much lower than that in the mdtR disruptant. These findings indicate that the MdtR protein is a repressor of the mdtRP operon and that the MdtP protein functions as a multidrug efflux transporter in B. subtilis.