Adeno-associated viral (AAV) vectors are the most efficient in vivo gene transfer tools for gene therapy applications. Efforts have been made to translate encouraging results in small animal models to human patients. However, the need for large quantities of vector for clinical application remains a great challenge. Developing novel AAV vectors with enhanced infectivity may reduce the high vector dose requirement for many applications such as gene therapy for muscular dystrophy. Selective mutation of AAV capsid surface-exposed tyrosine (Y) is a novel strategy to improve transduction efficiency. AAV6 has been considered one of the most robust muscle gene delivery vehicles. Here, we hypothesize that AAV6 transduction efficiency can be further enhanced by mutating surface Y to phenylalanine (F). We found that mutants AAV6-Y445F and AAV6-Y731F, especially the former, achieved more efficient gene transfer than the original AAV6 after intramuscular administration to mice. Expression of both firefly luciferase and alkaline phosphatase reporter genes increased up to 8-fold and DNA copy numbers in muscle increased up to 6-fold. Our results suggest that tyrosine-mutant AAV6 vectors may represent powerful tools for testing muscle gene therapy in animal models and potentially in humans.