Despite compelling evidence supporting key roles for glycogen synthase kinase 3β (GSK3β), mitochondrial adenosine triphosphate-sensitive K ⁺ (mitoK _ATP ) channels, and mitochondrial connexin 43 (Cx43) in cytoprotection, it is not clear how these signaling modules are linked mechanistically. By patch-clamping the inner membrane of murine cardiac mitochondria, we found that inhibition of GSK3β activated mitoK _ATP . PKC activation and protein phosphatase 2a inhibition increased the open probability of mitoK _ATP channels through GSK3β, and this GSK3β signal was mediated via mitochondrial Cx43. Moreover, ( i ) PKC-induced phosphorylation of mitochondrial Cx43 was reduced in GSK3β-S9A mice; ( ii ) Cx43 and GSK3β proteins associated in mitochondria; and ( iii ) SB216763-mediated reduction of infarct size was abolished in Cx43 KO mice in vivo, consistent with the notion that GSK3β inhibition results in mitoK _ATP opening via mitochondrial Cx43. We therefore directly targeted mitochondrial Cx43 by the Cx43 C-terminal binding peptide RRNYRRNY for cardioprotection, circumventing further upstream pathways. RRNYRRNY activated mitoK _ATP channels via Cx43. We directly recorded mitochondrial Cx43 channels that were activated by RRNYRRNY and blocked by the Cx43 mimetic peptide ⁴³ GAP27. RRNYRRNY rendered isolated cardiomyocytes in vitro and the heart in vivo resistant to ischemia/reperfusion injury, indicating that mitochondrial Cx43- and/or mitoK _ATP -mediated reduction of infarct size was not undermined by RRNYRRNY-related opening of sarcolemmal Cx43 channels. Our results demonstrate that GSK3β transfers cytoprotective signaling through mitochondrial Cx43 onto mitoK _ATP channels and that Cx43 functions as a channel in mitochondria, being an attractive target for drug treatment against cardiomyocyte injury.