Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca(2+). These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.