Increased hexosamine biosynthesis pathway (HBP) flux and elevated levels of protein O-linked-N-acetylglucosamine (O-GlcNAc) decrease calcium influx into isolated cardiomyocytes. Increased O-GlcNAc levels also increase tolerance of cells to stress. Therefore, the goal of this study was to test the hypothesis that increasing HBP flux and protein O-GlcNAc levels in the intact heart will increase the tolerance to tissue injury resulting from the calcium paradox and ischemia. We used two strategies that have been shown to increase HBP flux in the intact heart, namely a brief period of streptozotocin-induced diabetes and acute pretreatment of the isolated perfused heart with glucosamine. Isolated perfused rat hearts were exposed to the calcium paradox or to ischemia and reperfusion. Both diabetes and glucosamine significantly improved recovery in the isolated perfused rat heart following the calcium paradox with left ventricular developed pressure (LVDP) returning to ∼80% of baseline compared to 0% in controls (P < 0.05), and lactate dehydrogenase release being reduced by approximately fivefold (P < 0.05). In the diabetic group, azaserine, which inhibits the HBP, restored the sensitivity to the calcium paradox. Glucosamine treatment also improved functional recovery following ischemia/reperfusion (LVDP: 47 ± 9% vs. 95 ± 4%, P < 0.05) and this was associated with a threefold increase in O-GlcNAc levels (P < 0.05). Alloxan, an inhibitor of O-GlcNAc-transferase, blocked both the protection seen with glucosamine and the increase in O-GlcNAc. These data demonstrate that activation of the HBP with glucosamine may be a novel strategy for inducing cardioprotection, and that this appears to be mediated by an increase in protein O-GlcNAc levels. © 2005 Elsevier Ltd. All rights reserved.