Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP‐activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the α2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC‐1, a mitochondrial membrane potential‐sensitive dye, and laser confocal microscopy. In contrast, experiments with di‐8‐ANEPPS, a sarcolemmal‐potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning‐induced shortening of the action membrane potential. The preconditioning‐induced activation of sarcolemmal K_ATP channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal K_ATP channels opening, inhibited preconditioning‐induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning‐induced recruitment of sarcolemmal K_ATP channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal K_ATP channels without being a part of signaling pathway that regulates mitochondrial membrane potential. J. Cell. Physiol. 210: 224–236, 2007. © 2006 Wiley‐Liss, Inc.