Connexins are transmembrane proteins whose best known function is to form gap junction channels. Ventricular cardiomyocytes express the connexin isoform Cx43 and are rich in gap junctions essential for the normal propagation of the action potential. In addition to this physiological role, cardiomyocyte gap junctions contribute to the pathophysiology of ischaemia-reperfusion injury, mainly by synchronizing the onset of rigour contracture during ischaemia and cell-to-cell propagation of hypercontracture during reperfusion. More recently, it has been recognized that Cx43 plays a role in protection during ischaemic and pharmacological preconditioning and that this role is independent from gap junction-mediated communication. It was demonstrated that Cx43 is localized in cardiomyocyte mitochondria, at least in part in the inner mitochondrial membrane, where it is imported by the general mitochondrial membrane translocase system. Interfering with this import system or genetic ablation of Cx43 abolishes diazoxide-induced protection, indicating that mitochondrial Cx43 participates in the preconditioning cascade. The role of mitochondrial Cx43 in preconditioning appears to be related to reactive oxygen species signalling, but its molecular mechanisms have not been elucidated in detail. The present article reviews available evidence on the localization of Cx43 in cardiomyocyte mitochondria, its role in protection by preconditioning, and the potential molecular mechanisms involved. These data may help to understand the pathophysiology of myocardial ischaemia-reperfusion and ischaemic preconditioning and to identify new strategies for cardioprotection, and they may be particularly relevant to situations such as aging in which total and mitochondrial Cx43 contents have been shown to be reduced.