Oxygen sensing and redox signaling significantly affect bacterial physiology and host-pathogen interaction. Here we show that a Staphylococcus aureus two-component system, AirSR (anaerobic iron-sulfur cluster-containing redox sensor regulator, formerly YhcSR), responds to oxidation signals (O-2, H2O2, NO, etc) by using a redox-active [2Fe-2S] cluster in the sensor kinase AirS. Mutagenesis studies demonstrate that the [2Fe-2S] cluster is essential for the kinase activity of AirS. We have also discovered that a homologue of IscS (SA1450) in S. aureus is active as a cysteine desulfurase, which enables the in vitro reconstitution of the [2Fe-2S] cluster in AirS. Phosphorylation assays show that the oxidized AirS with a [2Fe-2S](2+) cluster is the fully active form of the kinase but not the apo-AirS nor the reduced AirS possessing a [2Fe-2S](+) cluster. Overoxidation by prolonged exposure to O-2 or contact with H2O2 or NO led to inactivation of AirS. Transcriptome analysis revealed that mutation of airR impacts the expression of similar to 355 genes under anaerobic conditions. Moreover, the mutant strain displayed increased resistance toward H2O2, vancomycin, norfloxacin, and ciprofloxacin under anaerobic conditions. Together, our results show that S. aureus AirSR is a redox-dependent global regulatory system that plays important roles in gene regulation using a redox active Fe-S cluster under O-2-limited conditions.