ABSTRACTStaphylococcus aureusis a pathogenic bacterium with a widespread distribution that can cause diverse severe diseases. The membrane-bound nitrate reductase NarGHJI serves respiratory function. However, little is known about its contribution to virulence. In this study, we demonstrated thatnarGHJIdisruption results in the downregulation of virulence genes (e.g.,RNAIII,agrBDCA,hla,psmα, andpsmβ) and reduces the hemolytic activity of the methicillin-resistantS. aureus(MRSA) strain USA300 LAC. Moreover, we provided evidence that NarGHJI participates in regulating host inflammatory response. A mouse model of subcutaneous abscess andGalleria mellonellasurvival assay demonstrated that theΔnarGmutant was significantly less virulent than the wild type. Interestingly, NarGHJI contributes to virulence in anagr-dependent manner, and the role of NarGHJI differs between differentS. aureusstrains. Our study highlights the novel role of NarGHJI in regulating virulence, thereby providing a new theoretical reference for the prevention and control ofS. aureusinfection.IMPORTANCEStaphylococcus aureusis a notorious pathogen that poses a great threat to human health. The emergence of drug-resistant strains has significantly increased the difficulty of preventing and treatingS. aureusinfection and enhanced the pathogenic ability of the bacterium. This indicates the importance of identifying novel pathogenic factors and revealing the regulatory mechanisms through which they regulate virulence. The nitrate reductase NarGHJI is mainly involved in bacterial respiration and denitrification, which can enhance bacterial survival. We demonstrated thatnarGHJIdisruption results in the downregulation of theagrsystem andagr-dependent virulence genes, suggesting that NarGHJI participates in the regulation ofS. aureusvirulence in anagr-dependent manner. Moreover, the regulatory approach is strain specific. This study provides a new theoretical reference for the prevention and control ofS. aureusinfection and reveals new targets for the development of therapeutic drugs.