Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. Polymyxins constitute an important class of antibiotics for treatment of infections caused by Gram-negative pathogens, whereas Gram-positive bacteria remain largely insusceptible towards class of antibiotics. Here we performed a whole-genome screen among nonessential genes for polymyxin intrinsic resistance determinants in Staphylococcus aureus We found that the ATP synthase is important for polymyxin susceptibility and that inhibition of the ATP synthase sensitizes S. aureus towards polymyxins. Our study provides novel insights into the mechanisms that limit polymyxin activity against S. aureus and provides valuable targets for inhibitors to potentially enable the use of polymyxins against S. aureus and other Gram-positive pathogens.