Transcriptional profiling of Staphylococcus aureus treated with cell wall-active antibiotics identified the 2-component system, VraSR, as one of the key players in response to antibiotic stress. Although it has been shown that a number of genes are regulated by the VraSR system, it has not been shown which genes are under direct VraSR regulation and which genes are not. In this study, chromatin immunoprecipitation techniques were used to identify the genes which are regulated by the direct interaction of VraR with their promoter regions. The results showed for the first time, that the VraSR mediated regulation of cell wall biosynthesis-associated genes, pbp2, murZ, and sgtB are facilitated by the direct binding of VraR to their respective promoters. Conversely, fmtA, indicated previously to be under VraSR regulation did not exhibit direct regulation by the binding of VraR to its promoter. The VraSR system plays a very important role in antibiotic resistance against cell wall-active antibiotics, and hence, it is essential to understand its complete regulatory mechanism.