The molecular mechanisms regulating skeletal muscle regeneration and differentiation are not well understood. We analyzed the expression of connexins (Cxs) 40, 43 and 45 in normal and regenerating tibialis anterior muscle and in primary cultures of differentiating myoblasts in adult and newborn mice, respectively. Cxs 45 and 43, but not 40, were strongly expressed in normal muscle and their expression was upregulated during regeneration. Furthermore, the functional role of Cx43 during differentiation and regeneration was examined after induced deletion of Cx43 in transgenic mice. In vivo, the inducible deletion of Cx43 delayed the formation of myofibers and prolonged the expression of myogenin during regeneration. In primary cultures of satellite cell-derived myoblasts, induced deletion of Cx43 led to decreased expression of myogenin and MyoD, dye coupling, creatine kinase activity and myoblast fusion. Thus, the expression of Cx45 and Cx43 is upregulated during skeletal muscle regeneration and Cx43 is required for normal myogenesis in vitro and adult muscle regeneration in vivo.