Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (DeltaAB/R3-R18/DeltaCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (DeltaAB/R3-R18/DeltaCT and DeltaR4-R23/DeltaCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of DeltaAB/R3-R18/DeltaCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized DeltaAB/R3-R18/DeltaCT. However, codon-optimized microdystrophin DeltaR4-R23/DeltaCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.