Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, a common cause of viral encephalitis in Asia. Here, we employed immunoprecipitation (IP) coupled with mass spectrometry (LC-MS/MS) to identify Myosin heavy chain 9 (MYH9) as a membrane protein that interacts with JEV virions in U251 cells. Immunofluorescence and IP revealed extensive co-localization of MYH9 with JEV virions during early infection. Knockout of MYH9 significantly decreased JEV replication as indicated by reduced progeny viral titers (about 31.6-fold in U251 cells, 11.7-fold in A549 cells), viral RNA abundance (about 98 % in U251 cells) and ‌JEV capsid (C) protein‌ abundance in both U251 and A549 cells. Conversely, MYH9 overexpression significantly enhanced JEV replication in both cells. Furthermore, MYH9 knockout significantly decreased JEV binding (about 61 %) and entry (about 35 %) in U251 cells. Antibodies and recombinant protein against MYH9 significantly inhibited JEV binding (about 43 % at 10 μM protein, 24 % at 24 μg/ml antibodies), entry (about 27 % at 10 μM protein, 22 % reduction at 24 μg/ml antibodies), and replication (about 13.5-fold in progeny viral titers at 10 μM protein, 5.1-fold at 24 μg/ml antibodies) in U251 cells. IP, Co-IP and ELISA showed the PRA domain of MYH9 interacted with JEV envelope (E) protein domain I-II. The MYH9 inhibitor blebbistatin significantly inhibited JEV infection in vitro and provided partial protection against JEV infection in vivo. Our results indicate that MYH9 is a novel host factor for JEV entry and replication and could be a potential therapeutic target.Copyright © 2025. Published by Elsevier B.V.