We have previously shown that pre-ischemic treatment with glucosamine improved cardiac functional recovery following ischemia/reperfusion (I/R) and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway (HBP) and subsequently elevated protein O-GlcNAc levels. However, pre-ischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min global, no flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10mM) or an inhibitor of O-GlcNAcase O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc, 200µM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared to untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of -fodrin as well as Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) during reperfusion. Thus, two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due at least in part to inhibition of calcium-mediated stress responses.