P otassium (K⁺) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoK_ATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoK_ATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide ⁴³GAP27 each substantially reduced diazoxide-mediated stimulation of mitoK_ATP channels. Suppression of mitochondrial Cx43 inhibited mitoK_ATP channel activation by PKC. MitoK_ATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoK_ATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoK_ATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.