The insulin-like family of factors are involved in the regulation of a variety of physiological processes, but the function of the family member termed insulin-like 6 (Insl6) in skeletal muscle has not been reported. We show that Insl6 is a myokine that is up-regulated in skeletal muscle downstream of Akt signaling and in regenerating muscle in response to cardiotoxin (CTX)-induced injury. In the CTX injury model, myofiber regeneration was improved by the intramuscular or systemic delivery of an adenovirus expressing Insl6. Skeletal muscle-specific Insl6 transgenic mice exhibited normal muscle mass under basal conditions but elevated satellite cell activation and enhanced muscle regeneration in response to CTX injury. The Insl6-mediated regenerative response was associated with reductions in muscle cell apoptosis and reduced serum levels of creatine kinase M. Overexpression of Insl6 stimulated proliferation and reduced apoptosis in cultured myogenic cells. Conversely, knockdown of Insl6 reduced proliferation and increased apoptosis. These data indicate that Insl6 is an injury-regulated myokine that functions as a myogenic regenerative factor.