It has been shown that orally administered geranylgeranylacetone (GGA), an anti-ulcer drug, induces expression of heat shock protein 72 (HSP72) and provides protection against ischemia/reperfusion in rat hearts. The underlying protective mechanisms, however, remain unknown. Mitochondria have been shown to be a selective target for heat-stress (HS)-induced cardioprotection. Therefore, we hypothesized that preservation of mitochondrial function, owing to opening of a putative channel in the inner mitochondrial membrane, the mitochondrial adenosine-triphosphate (ATP)-sensitive potassium (mitoK_ATP) channel, could be involved in GGA- or HS-induced cardioprotection against ischemia/reperfusion. Rats were treated with oral GGA or vehicle. Twenty-four hours later, each heart was isolated and perfused with a Langendorff apparatus. GGA-treated hearts showed better functional recovery and less creatine kinase was released during a 30-minute reperfusion period, after 20 minutes of no-flow ischemia. Concomitant perfusion with 5-hydroxydecanoate (5HD, 100 µM) or glibenclamide (10 µM) abolished the GGA-induced cardioprotective effect. GGA also showed preserved mitochondrial respiratory function, isolated at the end of the reperfusion period, which was abolished with 5HD treatment. GGA prevented destruction of the mitochondrial structure by ischemia/reperfusion, as shown by electron microscopy. In cultured cardiomyocytes, GGA induced HSP72 expression and resulted in less damage to cells, including less apoptosis in response to hypoxia/reoxygenation. Treatment with 5HD abolished the GGA-induced cardioprotective effects, but did not affect HSP72 expression. Our results indicate that preserved mitochondrial respiratory function, owing to GGA-induced HSP72 expression, may, at least in part, have a role in cardioprotection against ischemia/reperfusion. These processes may involve opening of the mitoK_ATP channel.