Recombinant adeno-associated virus (rAAV) vectors are one of the most promising in vivo gene delivery tools. Several features make rAAV vectors an ideal platform for gene transfer. However, the high homology with the parental wild-type virus, which often infects humans, poses limitations in terms of immune responses associated with this vector platform. Both humoral and cell-mediated immunity to wild-type AAV have been documented in healthy donors, and, at least in the case of anti-AAV antibodies, have been shown to have a potentially high impact on the outcome of gene transfer. While several factors can contribute to the overall immunogenicity of rAAV vectors, vector design and the total vector dose appear to be responsible of immune-mediated toxicities. While preclinical models have been less than ideal in predicting the outcome of gene transfer in humans, the current preclinical body of evidence clearly demonstrates that rAAV vectors can trigger both innate and adaptive immune responses. Data gathered from clinical trials offers key learnings on the immunogenicity of AAV vectors, highlighting challenges as well as the potential strategies that could help unlock the full therapeutic potential of in vivo gene transfer.