In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to < 30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.