Staphylococcus aureus is a major human pathogen that causes diverse severe infections. The emergence of multi-drug resistance has caused S. aureus infection a global concern, especially resistance to vancomycin, a drug of last resort. A growing body of research focuses on the resistance mechanisms and antibiotic discovery. S-nitrosylation, a posttranslational modification on cysteine thiol mediated by Nitric oxide (NO) has been found to drive a large part of the ubiquitous influence of NO on mediating cellular functions. Several gram-positive microorganisms, including S. aureus, possess a bacterial NO synthase (bNOS) to generate NO endogenously. bNOS was reported to play an important role in protecting bacteria against a wide spectrum of antibiotics, but the precise underlying mechanism remains elusive. Here, S-nitrosylation patterns were analyzed in a clinically isolated vancomycin-intermediate staphylococcus aureus strain XN108 by liquid chromatography-mass spectrometry (LC-MS)/MS. The MS data of samples XN_1 and XN_2 were integrated and analyzed.