Irreversible myocardial injury following ischemia/reperfusion is reduced by ischemic or pharmacological pre- and postconditioning. The different signal transduction cascades activated by such cardioprotective interventions converge at the level of the mitochondria, where they induce opening of mitochondrial ATP-dependent potassium channels and inhibition of mitochondrial permeability transition pore (MPTP) opening. Connexin 43 (Cx43) is present at gap junctions but also at the inner membrane of cardiomyocyte mitochondria. Mitochondrial Cx43 regulates respiration and generation of reactive oxygen species. In addition, Cx43 contributes to diazoxide-induced opening of mitochondrial ATP-dependent potassium channels. The specific reduction of mitochondrial Cx43 is sufficient to attenuate infarct size reduction by pharmacological preconditioning with diazoxide. The signal transducer and activator of transcription 3 (STAT3) is part of the janus kinase/STAT signal transduction pathway and regulates the transcription of target genes in the nucleus. STAT3 is also present in the matrix of cardiomyocyte mitochondria. Genetic ablation and pharmacological inhibition of mitochondrial STAT3 reduce oxygen consumption and enhance calcium-induced MPTP opening. STAT3 co-immunoprecipitates with cyclophilin D, the target of the cardioprotective agent and MPTP inhibitor cyclosporine A (CsA). However, CsA reduces infarct size to a similar extent in wildtype and STAT3-deficient mice in vivo. In contrast, ischemic postconditioning is dependent on the presence of STAT3, since ischemic postconditioning does not reduce infarct size in STAT3-deficient mice. Taken together, our data demonstrate that Cx43 and STAT3 impact on mitochondrial function and that preservation of the amount of mitochondrial Cx43 and STAT3 is important for the cardioprotection by ischemic pre- and postconditioning.