Rabies virus (RABV) invades the central nervous system and nearly always causes fatal disease in humans. RABV enters cells via clathrin-mediated endocytosis upon receptor binding. The detailed mechanism of this process and how it is regulated are not fully understood. Here, we carried out a high-through-put RNAi analysis and identified AP2-associated kinase 1 (AAK1), a serine/threonine kinase, as an important cellular component in regulating the entry of RABV. AAK1 knock-down greatly inhibits RABV infection of cells, and AAK1-induced phosphorylation of threonine 156 of the &mu / subunit of adaptor protein 2 (AP2M1) is found to be required for RABV entry. Inhibition of AAK1 kinase activity by sunitinib blocked AP2M1 phosphorylation, significantly inhibiting RABV infection and preventing RABV from entering early endosomes. In vivo studies revealed that sunitinib prolongs the survival of mice challenged with RABV street virus. Our findings indicate that AAK1 is a potential drug target for postexposure prophylaxis against rabies.