This study aims to determine the effect of exercise on the cardiac function, metabolic profiles and related molecular mechanisms in mice with ischemic-induced heart failure (HF). HF was induced by myocardial infarction (MI) in C57BL6/N mice. Cardiac function and physical endurance were improved in HF mice after exercise. Micro-PET/CT scanning revealed enhanced myocardial glucose uptake in vivo in HF mice after exercise. Exercise reduced mitochondrial structural damage in HF mice. Cardiomyocytes isolated from HF + exercise mice showed increased glycolysis capacity, respiratory function and ATP production. Both mRNA and protein expression of glucose transporter 1 (GLUT1) were upregulated after exercise. Results of ChIP-PCR revealed a novel interaction between transcription factor myocyte enhancer factor 2a (MEF2a) and GLUT1 in hearts of HF + exercise mice. Exercise also activated myocardial AMP-activated protein kinase (AMPK), which in turn phosphorylated histone deacetylase 4 (HDAC4), and thereby modulated the GLUT1 expression through reducing its inhibition on MEF2a in HF mice. Inhibition of HDAC4 also improved cardiac function in HF mice. Moreover, knockdown of GLUT1 impaired the systolic and diastolic function of isolated cardiomyocytes. In conclusion, exercise improves cardiac function and glucose metabolism in HF mice through inhibiting HDAC4 and upregulating GLUT1 expression.