Pompe disease results from acid α-glucosidase (GAA) deficiency and enzyme replacement therapy (ERT) with recombinant human (rh) GAA has clinical benefits, although its limitations include the short half-life of GAA and the formation of antibody responses. The present study compared the efficacy of ERT against gene transfer with an adeno-associated viral (AAV) vector containing a liver-specific promoter. GAA knockout (KO) mice were administered either a weekly injection of rhGAA (20 mg/kg), or a single injection of AAV2/8-LSPhGAA (8x1011 vector genomes (vg)/kg). Both treatments significantly reduced glycogen content of the heart and diaphragm. While ERT triggered anti-GAA antibody formation, there was no detectable antibody response following AAV vector administration. The efficacy of 3 lower dosages of AAV2/8-LSPhGAA was evaluated in GAA-KO mice, either alone or in combination with ERT. The minimum effective dose (MED) identified was 8x1010 vg/kg to reduce glycogen content in the heart and diaphragm of GAA-KO mice. A 3-fold higher dose was required to suppress antibody responses to ERT. Efficacy from liver gene therapy was slightly greater in male mice than in females. Vector dose correlated inversely with anti-GAA antibody formation, while higher vector doses suppressed previously formed anti-GAA antibodies as late as 25 weeks after the start of ERT and achieved biochemical correction of glycogen accumulation. In conclusion, we identified the MED for effective AAV2/8-LSPhGAA-mediated tolerogenic gene therapy in Pompe disease mice.