Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H2O2). Here we show that H2O2 reactivity of the active site thiolate (C152) is catalyzed by a previously unrecognized mechanism based on a dedicated proton relay promoting leaving group departure. Disruption of the peroxidatic reaction mechanism does not affect the glycolytic activity of GAPDH. Therefore, specific and separate mechanisms mediate the reactivity of the same thiolate nucleophile toward H2O2 and glyceraldehyde 3-phosphate, respectively. The generation of mutants in which the glycolytic and peroxidatic activities of GAPDH are comprehensively uncoupled allowed for a direct assessment of the physiological relevance of GAPDH H2O2 sensitivity. Using yeast strains in which wild-type GAPDH was replaced with H2O2-insensitive mutants retaining full glycolytic activity, we demonstrate that H2O2 sensitivity of GAPDH is a key component of the cellular adaptive response to increased H2O2 levels.