Updated project metadata. Redox signaling and cardiac function are tightly linked. Still, it is largely unknown which specific protein targets are affected by reactive oxygen species (ROS) that underly the impaired inotropic effect in oxidative stress. Here, we combined a new chemogenetic mouse model (HMC HyPer-DAO transgenic mice) and a redox proteomics approach to identify cellular redox switches and their functional role. Using the HyPer-DAO mice, we prove that increased endogenous production of H2O2 in cardiomyocytes is leading to a reversible cardiac contractility in vivo. We identified the -subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch and linked this modification to mitochondrial metabolism and glutathione synthesis. Molecular dynamics simulations combined with experimental evidence from cysteine-gene-edited point mutations revealed that IDH3 Cys148 and 284 are critically involved in oxidant-dependent alterations of IDH3 function. Together, our results demonstrate a specific link between ROS, IDH3 and mitochondrial metabolism. Cardiac phenotyping of the chemogenetic mice reveal the biological significance of these ROS-induced modifications.