Reflexes initiated by the carotid body, the principal O₂-sensing organ, are critical for maintaining cardiorespiratory homeostasis during hypoxia. O₂ sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H₂S) synthesis by cystathionine-γ-lyase (CSE). We report that O₂ stimulated the generation of CO, but not that of H₂S, and required two cysteine residues in the heme regulatory motif (Cys²⁶⁵ and Cys²⁸²) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser³⁷⁷ of CSE, inhibiting the production of H₂S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H₂S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O₂ sensing through PKG-dependent regulation of H₂S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.