Activation of melatonin receptors induces cardioprotection. Mitochondrial potassium channels (mKCa and mKATP) are involved in the signaling cascade of preconditioning. The melatonin receptor agonist ramelteon is an approved oral medication for treatment of insomnia, but nothing is known about possible cardioprotective properties. We investigated whether (1) ramelteon induces cardioprotection mediated by the melatonin receptor; (2) this effect is concentration-dependent; and (3) mKCa and/or mKATP channels are critically involved in ramelteon-induced cardioprotection. Hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mm Hg. All hearts were subjected to 33 minutes of global ischemia and 60 minutes of reperfusion. Before, ischemic hearts were perfused with different concentrations of ramelteon (0.01-5 μM) for determination of a concentration-effect curve. In subsequent experiments, the lowest protective concentration of ramelteon was administered together with paxilline (mKCa channel inhibitor) and 5-hydroxydecanoate (mKATP channel inhibitor). To determine whether the reduction of ischemia and reperfusion injury by ramelteon is mediated by melatonin receptor, we combined ramelteon with luzindole, a melatonin receptor antagonist. Infarct size was determined by triphenyltetrazolium chloride staining. In control animals, infarct size was 58% ± 6%. Ramelteon in a concentration of 0.03 µM reduced infarct size to 28% ± 4% (P < 0.0001 vs. Con). A lower concentration of ramelteon did not initiate cardioprotection, and higher concentrations did not further decrease infarct size. Paxilline, 5-hydroxydecanoate, and luzindole completely blocked the ramelteon-induced cardioprotection. This study shows for the first time that (1) ramelteon induces cardioprotection through melatonin receptor; (2) the effect is not concentration-dependent; and (3) activation of mKCa and mKATP channels is involved.