Physiological sensing of O₂ tension (partial O₂ pressure, pO₂) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO₂ to altered calcium release through the ryanodine receptor–Ca²⁺-release channel (RyR1). Reactive oxygen species are generated in proportion to pO₂ by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca²⁺ release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O₂ sensor in skeletal muscle, and O₂-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O₂-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.