Immune responses against AAV are considered a major challenge in AAV gene therapy. We aimed to reduce the innate immune recognition of AAV vectors by interfering with Toll-like receptor signaling. Using capsid engineering, we inserted a myeloid differentiation primary response 88 (MyD88)- derived peptide that is capable of blocking MyD88 dimerization. Insertion of the MyD88 peptide into the capsid did not hamper capsid formation or vector production yield. The new capsid variant, AAV2.MB453, demonstrated enhanced transduction compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. Furthermore, in primary human cells reduced innate immune responses, including type I interferons, were observed compared to AAV2. In vivo, reduced CD8+ T cell responses against the transgene product and against the capsid were observed for AAV2.MB453. Furthermore, AAV2.MB453 treated mice showed delayed generation of AAV2-binding IgG2a antibodies and an increased Nab50. In summary, by inserting a MyD88-derived peptide into the AAV2 capsid we developed a novel variant with improved transduction efficiency and lower innate and adaptive immune responses.