<p>Myocardial ischemia/reperfusion (I/R) injury poses a significant worldwide health burden. O-Linked β-N-acetylglucosamine transferase (OGT), an enzyme that catalyzes protein O-GlcNAcylation, is implicated in cellular stress responses, but its precise role and mechanism in cardioprotection are not fully elucidated. In this study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury, and cardiomyocyte injury was evaluated by assessing cell viability, lactate dehydrogenase (LDH) release, apoptosis, and oxidative stress markers. In parallel, mice were pretreated with the OGT activator Thiamet-G or the OGT inhibitor OSMI-1 prior to left anterior descending artery ligation, followed by comprehensive evaluation of cardiac function, myocardial injury biomarkers, and histopathological changes. We found that OGT overexpression markedly attenuated H/R-induced cardiomyocyte injury, as evidenced by improved cell viability, reduced LDH release and apoptosis, and alleviated oxidative stress. Consistently, pharmacological activation of OGT improved cardiac function and reduced myocardial fibrosis following I/R injury in vivo, whereas OGT inhibition exerted the opposite effect. Mechanistically, co-immunoprecipitation, western blotting, and site-directed mutagenesis revealed that OGT enhances global protein O-GlcNAcylation and increases the stability of cardioprotective protein MG53 by catalyzing its O-GlcNAcylation at the Ser301 residue, and the protective effects of OGT were abolished in cardiomyocytes expressing the MG53 Ser301 mutant. Collectively, these findings demonstrate that MG53 is a key downstream substrate of OGT-mediated O-GlcNAcylation in myocardial I/R injury and provide mechanistic insight into how OGT confers cardioprotection, highlighting the OGT–MG53 axis as a potential therapeutic target for mitigating cardiac I/R injury.</p>