The mechanisms that regulate skeletal muscle differentiation, fiber type diversity and muscle regeneration are incompletely defined. Forkhead transcription factors are critical regulators of cellular fate determination, proliferation, and differentiation. We identified a forkhead/winged helix transcription factor, Foxj3, which was expressed in embryonic and adult skeletal muscle. To define the functional role of Foxj3, we examined Foxj3 mutant mice. Foxj3 mutant mice are viable but have significantly fewer Type I slow-twitch myofibers and have impaired skeletal muscle contractile function compared to their wild type controls. In response to a severe injury, Foxj3 mutant mice have impaired muscle regeneration. Foxj3 mutant myogenic progenitor cells have perturbed cell cycle kinetics and decreased expression of Mef2c. Examination of the skeletal muscle 5′ upstream enhancer of the Mef2c gene revealed an evolutionary conserved forkhead binding site (FBS). Transcriptional assays in C2C12 myoblasts revealed that Foxj3 transcriptionally activates the Mef2c gene in a dose dependent fashion and binds to the conserved FBS. Together, these studies support the hypothesis that Foxj3 is an important regulator of myofiber identity and muscle regeneration through the transcriptional activation of the Mef2c gene.