Halogenated 4-hydroxybenzylidene indolinones have been shown to re-sensitize methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) to methicillin and vancomycin respectively. The mechanism of antibiotic re-sensitization was however not previously studied. Here, we probe the scope of antibiotic re-sensitization and present the global proteomics analysis of S. aureus treated with GW5074, a 4-hydroxybenzylidene indolinone compound. With a minimum inhibitory concentration (MIC) of 8 μg/mL against S. aureus, GW5074 synergized with beta-lactam antibiotics like ampicillin, carbenicillin and cloxacillin, the DNA synthesis inhibitor, ciprofloxacin, the protein synthesis inhibitor, gentamicin and the folate acid synthesis inhibitor, trimethoprim. Global proteomics analysis revealed that GW5074 treatment resulted in significant downregulation of enzymes involved in the purine biosynthesis. S. aureus proteins involved in amino acid metabolism and peptide transport were also observed to be downregulated. Interestingly, anti-virulence targets such as AgrC (a quorum sensing-related histidine kinase), AgrA (a quorum sensing-related response regulator) as well as downstream targets, such as hemolysins, lipases and proteases in S. aureus were also downregulated by GW5074. We observed that the peptidoglycan hydrolase, SceD was significantly upregulated. The activity of GW5074 on S. aureus suggests that the compound primes bacteria for the antibacterial action of ineffective antibiotics. SIGNIFICANCE: Antibiotic resistance continues to present significant challenges to the treatment of bacterial infections. Given that antibiotic resistance is a natural phenomenon and that it has become increasingly difficult to discover novel antibiotics, efforts to improve the activity of existing agents are worth pursuing. A few small molecules that re-sensitize resistant bacteria to traditional antibiotics have been described but the molecular details that underpin how these compounds work to re-sensitize bacteria remain largely unknown. In this report, global label-free quantitative proteomics was used to identify changes in the proteome that occurs when GW5074, a compound that re-sensitize MRSA to methicillin, is administered to S. aureus. The identification of pathways that are impacted by GW5074 could help identify novel targets for antibiotic re-sensitization.