Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8–12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8–12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.