Current treatment for serious infections caused by methicillin-resistant Staphylococcus aureus relies heavily upon the glycopeptide antibiotic vancomycin. Unfortunately, this practice has led to an intermediate resistance phenotype that is particularly difficult to treat in invasive staphylococcal diseases, such as septicemia and its metastatic complications, including endocarditis. Although the vancomycin-intermediate resistance phenotype has been linked to abnormal cell wall structures and autolytic rates, the corresponding genetic changes have not been fully elucidated. Previously, whole-genome array studies listed numerous genes that are overexpressed in vancomycin-intermediate sensitive strains, including graRS (SACOL0716 to -0717), encoding a two-component regulatory system (TCRS), as well as the adjacent vraFG (SACOL0718 to -0720), encoding an ATP-binding cassette (ABC) transporter; but the exact contribution of these genes to increased vancomycin resistance has not been defined. In this study, we showed that isogenic strains with mutations in genes encoding the GraRS TCRS and the VraFG ABC transporter are hypersensitive to vancomycin as well as polymyxin B. Moreover, GraRS regulates the expression of the adjacent VraFG pump, reminiscent of gram-positive bacteriocin-immunity regulons. Mutations of graRS and vraFG also led to increased autolytic rates and a more negative net surface charge, which may explain, in part, to their increased sensitivity to cationic antimicrobial peptides. Taken together, these data reveal an important genetic mediator to the vancomycin-intermediate S. aureus phenotype and may hold clues to the selective pressures on staphylococci upon exposure to selective cationic peptide antibiotics used in clinical practice.