Widespread anti-AAV antibodies (Abs) in humans pose a critical challenge for the translation of AAV gene therapies, limiting patient eligibility. In this study, non-human primates (NHPs) with pre-existing αAAV Abs were used to investigate the impact of αAAV9 Ab levels on the transduction efficiency of rAAV9 via systemic delivery. No significant differences were observed in vector genome (vg) biodistribution in animals with ≤1:400 total serum αAAV9-IgG compared to αAAV9-Ab-negative animals, following an intravenous (i.v.) rAAV9-h (codon-optimized human α-N-acetylglucosaminidase coding sequence cDNA) injection. Serum αAAV9-IgG at >1:400 resulted in a >200-fold decrease in vg in the liver, but had no significant effect on vg levels in brain and most of the peripheral tissues. Although tissue NAGLU activities declined significantly, they remained above endogenous levels. Notably, there were higher vg copies but lower NAGLU activity in the spleen in NHPs with >1:400 αAAV9 Abs than in those with ≤1:400 Abs. We demonstrate here the presence of a threshold of pre-existing αAAV9 Abs for diminishing the transduction of i.v.-delivered AAV vectors, supporting the expansion of patient eligibility for systemic rAAV treatments. Our data also indicate that high pre-existing αAAV9 Abs may promote phagocytosis and that phagocytized vectors are not processed for transgene expression, suggesting that effectively suppressing innate immunity may have positive impacts on transduction efficiency in individuals with high Ab titers.