Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 μM CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 μM CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA ≈2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.