Metallothionein (MT) is effective in the prevention of diabetic cardiomyopathy, and hypoxia-inducible factor-1 (HIF-1) is known to control vascular endothelial growth factor (VEGF) gene expression and regulate angiogenesis in diabetic hearts. We examined whether or not MT affects HIF-1 activity in the heart of diabetic mice and in the cardiac cells cultured in high glucose (HG) media. Diabetes was induced by streptozotocin in a cardiac-specific MT overexpressing transgenic mouse model. The primary cultures of neonatal cardiomyocytes and the embryonic rat cardiac H9c2 cell line were cultured in HG media. HIF-1 and VEGF were determined by immunofluorescent staining and enzyme-linked immunosorbent assay, respectively. The H9c2 cells were transfected with a hypoxia-responsive element-dependent reporter plasmid and the HIF-1 transcriptional activity was measured by luciferase reporter assay. MT overexpression increased HIF-1alpha in diabetic hearts. HG suppressed CoCl(2)-induced VEGF expression in primary cultures of neonatal cardiomyocytes and MT overexpression suppressed the inhibition. The addition of MT into the cultures of H9c2 cells relieved the HG suppression of hypoxia-induced luciferase activity. This study indicates that MT can rescue HIF-1 transcriptional activity in cardiomyocytes under diabetic conditions.