AIMS: Experimental evidence has shown significant cardioprotective effects of insulin, whereas clinical trials produced mixed results without valid explanations. This study was designed to examine the effect of hyperglycaemia on insulin cardioprotective action in a preclinical large animal model of myocardial ischaemia/reperfusion (MI/R). METHODS AND RESULTS: Anaesthetized dogs were subjected to MI/R (30 min/4 h) and randomized to normal plasma insulin/euglycaemia (NI/NG), normal-insulin/hyperglycaemia (NI/HG), high-insulin/euglycaemia (HI/NG), and high-insulin/hyperglycaemia (HI/HG) achieved by controlled glucose/insulin infusion. Endogenous insulin production was abolished by peripancreatic vessel ligation. Compared with the control animals (NI/NG), hyperglycaemia (NI/HG) significantly aggravated MI/R injury. Insulin elevation at clamped euglycaemia (HI/NG) protected against MI/R injury as evidenced by reduced infarct size, decreased necrosis and apoptosis, and alleviated inflammatory and oxidative stress (leucocyte infiltration, myeloperoxidase, and malondialdehyde levels). However, these cardioprotective effects of insulin were markedly blunted in hyperglycaemic animals (HI/HG). In vitro mechanistic study in neonatal rat cardiomyocytes revealed that insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt was significantly attenuated by high glucose, accompanied by markedly increased IRS-1 O-GlcNAc glycosylation following hypoxia/reoxygenation. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine abrogated high glucose-induced O-GlcNAc modification and inactivation of IRS-1/Akt as well as cell injury. CONCLUSIONS: Our results, derived from a canine model of MI/R, demonstrate that hyperglycaemia blunts insulin protection against MI/R injury via hyperglycaemia-induced glycosylation and subsequent inactivation of insulin-signalling proteins. Our findings suggest that prevention of hyperglycaemia is critical for achieving maximal insulin cardioprotection for the ischaemic/reperfused hearts.