Adeno-associated virus (AAV) infection begins with the virus binding to heparan sulfate proteoglycan at the surface of the target cell, but additional receptors are thought to be necessary for viral entry. Now, using a haploid genetic screen, Pillay et al. identify the type I transmembrane protein KIAA0319L as an essential receptor that mediates AAV entry and rename this protein the AAV receptor (AAVR). The function of AAVR was confirmed by using CRISPR–Cas9 to knock out the receptor and by using anti-AAVR blocking antibodies; both treatments rendered cells highly resistant to AAV infection with several serotypes, including AAV2, which is the most commonly used serotype for gene therapy in clinical trials. Finally, Aavr knockout mice were more resistant to AAV-mediated gene therapy than mice that expressed AAVR.