Background: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, resulting in α-galactosidase A (α-Gal A) deficiency and progressive accumulation of globotriaosylceramide (Gb3). Current therapies, such as enzyme replacement and chaperone therapy, have limitations, including incomplete biodistribution and mutation-specific efficacy. Gene therapy using adeno-associated virus (AAV) vectors presents a promising alternative. Methods: In this study, we assessed the dose-dependent effects of AAV8 and AAV9 vectors encoding human GLA in Gla knockout (Gla^−/y) mice by measuring α-Gal A activity and monitoring safety. To evaluate therapeutic efficacy, symptomatic Fabry mice (G3S^Tg/+Gla^−/y) were used. Results: AAV9-GLA produced significantly higher and more sustained enzyme activity than AAV8-GLA across plasma, liver, heart, and kidney. In symptomatic mice, AAV9-GLA achieved superior reductions in serum Gb3 and lyso-Gb3 levels, greater Gb3 clearance in heart and kidney tissues, and improved proteinuria. Anti-GLA IgG titers remained below threshold for the first four weeks and increased modestly by week eight, indicating a limited humoral immune response. No significant clinical signs or weight loss were observed in Gla^−/y mice over the 3.5-month study period, supporting the favorable safety profile of AAV-mediated gene therapy. Conclusions: These findings demonstrate that AAV9 provides enhanced biodistribution and therapeutic efficacy compared to AAV8, supporting its potential for the treatment of Fabry disease.