Autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD 2D) is caused by mutations in the alpha-sarcoglycan gene (alpha-SG). The absence of alpha-SG results in the loss of the SG complex at the sarcolemma and compromises the integrity of the sarcolemma. To establish a method for recombinant adeno-associated virus (rAAV)-mediated alpha-SG gene therapy into alpha-SG-deficient muscle, we constructed rAAV serotypes 2 and 8 expressing the human alpha-SG gene under the control of the ubiquitous cytomegalovirus promoter (rAAV2-alpha-SG and rAAV8-alpha-SG). We compared the transduction profiles and evaluated the therapeutic effects of a single intramuscular injection of rAAVs into alpha-SG-deficient (Sgca(-/-)) mice. Four weeks after rAAV2 injection into the tibialis anterior (TA) muscle of 10-day-old Sgca(-/-) mice, transduction of the alpha-SG gene was localized to a limited area of the TA muscle. On the other hand, rAAV8-mediated alpha-SG expression was widely distributed in the hind limb muscle, and persisted for 7 months without inducing cytotoxic and immunological reactions, with a reversal of the muscle pathology and improvement in the contractile force of the Sgca(-/-) muscle. This extensive rAAV8-mediated alpha-SG transduction in LGMD 2D model animals paves the way for future clinical application.