Defective glycolysis and angiogenesis in the heart of diabetic patients and in experimental diabetic animal models have been reported. The aim of this study was to determine whether overexpression of hypoxia-inducible factor (HIF)-1alpha protects from myocardial injury in diabetic mice by increasing myocardial glycolysis and angiogenesis. Cardiac-specific HIF-1alpha-overexpressing transgenic and age-matched wild-type control mice were treated with streptozotocin to induce diabetes. Changes in glucose transporters, glycolytic enzymes, angiogenic factors and cardiac morphology were examined in the hearts by real-time RT-PCR, Western blotting, enzymatic assay, and histological assays. HIF-1alpha overexpression elevated hexokinase II (HK-II) protein level and total HK activity in nondiabetic heart and prevented the decreases in HK-II mRNA, protein, and total HK activity in diabetic heart. In addition, the reduction of glucose transporter I, but not glucose transporter 4, was restored in HIF transgenic mouse heart along with a recovery of myocardium ATP production. HIF-1alpha overexpression also normalized diabetes-reduced vascular endothelial growth factor concentration along with a sustained myocardial capillary density and an inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. Therefore, elevation of HIF-1alpha provides a cardiac protection from diabetic-induced impairment in glucose metabolism and angiogenesis via up-regulation of HIF-1 target genes.