During ischemia and heart failure there is an increase in cardiac glycolysis. To understand if this is beneficial or detrimental to the heart we chronically elevated glycolysis by cardiac-specific overexpression of phosphatase deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) in transgenic mice. PFK-2 controls the level of fructose-2,6-bisphosphate (Fru-2,6-P₂), an important regulator of phosphofructokinase and glycolysis. Transgenic mice had over a three-fold elevation in levels of Fru-2,6-P₂ . Cardiac metabolites upstream of phosphofructokinase were significantly reduced as would be expected by activation of phosphofructokinase. In perfused hearts the transgene caused a significant increase in glycolysis which was resistant to normal regulation by palmitate. The elevation in glycolysis made isolated cardiomyocytes highly resistant to contractile inhibition by hypoxia but in vivo the transgene had no effect on ischemia-reperfusion injury. Transgenic hearts exhibited pathology: heart to body weight was increased 17%, cardiomyocyte length was greater and cardiac fibrosis was increased. The results show that elevation in glycolysis provides acute benefits against hypoxia, but the chronic increase in glycolysis interferes with normal cardiac metabolic regulatory mechanisms which may be detrimental to the heart.