The stimulation of glucose transport by metabolic stress is an important determinant of myocardial susceptibility to ischemia and reperfusion injury. Stimulation of glucose transport is markedly impaired in cardiomyocytes chronically exposed to excess free fatty acids (FFA), as occurs in vivo in type 2 diabetes. To determine whether chronic low-grade activation of AMP-activated kinase (AMPK) improves substrate metabolism in cardiomyocytes exposed to FFA, isolated cultured cardiomyocytes were exposed for 7 days to FFA ± the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Glucose transport and glycolysis were then measured during acute metabolic stress provoked by oligomycin. Chronic treatment with AICAR improved basal and oligomycin-stimulated glucose transport in FFA-exposed but not in control cardiomyocytes. Similarly, basal and oligomycin-stimulated glycolysis was reduced in FFA-exposed cardiomyocytes but restored by chronic AICAR treatment. Conversely, fatty acid oxidation was increased in FFA-exposed cardiomyocytes and reduced by chronic AICAR treatment. Chronic AICAR treatment induced in FFA-exposed cardiomyocytes the biogenesis of numerous lipid droplets. Curiously, whereas acute treatment of cardiomyocytes with AICAR increased phosphorylation of the AMPKα subunit on T172, a classical marker of AMPK activation, chronic AICAR treatment almost completely obliterated T172 phosphorylation. However, phosphorylation of the AMPK target protein raptor on S792 was reduced in FFA-exposed cardiomyocytes but restored by AICAR treatment. In conclusion, chronic AICAR treatment induces a metabolic shift in FFA-exposed cardiomyocytes, characterized by improved glucose transport and glycolysis and redirection of fatty acids towards neutral storage. Such metabolic changes in vivo could protect the hearts of patients with type 2 diabetes against ischemia-reperfusion injury.