Activation of AMPK may benefit the heart during ischemia/reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative AMPK₂ subunit (AMPK Dn) to 20 min global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-¹⁴C]glucose, 0.4 mM [9, 10-³H]palmitate, and 100 µU/ml insulin. The AMPK Dn hearts had reduced AMPK activity at the end of reperfusion, (82±9 vs. 141±7 pmol/mg/min) with no changes in high-energy phosphates. Despite this, AMPK₂ Dn hearts had improved recovery of function during reperfusion (14.9±0.8 vs. 9.4±1.4 bpm•mmHg•10^-3). During reperfusion, fatty acid oxidation provided 52.6±6.4% of total acetyl-CoA in the AMPK Dn hearts compared to 55.0±3.2% in the control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups, despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4±9.4% vs. 85.3±4.3%). These data demonstrate that suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function following ischemia.