Infectious pancreatic necrosis virus (IPNV) is a highly pathogenic agent of salmonid. However, the critical factors responsible for IPNV binding largely remain unknown. Here, by using a combination of immunoprecipitation and mass spectrometry assays, we identified that non-muscle myosin heavy chain 9 (Myh9) played an important role in IPNV entry by interacting with viral outer capsid protein VP2 on cell surface. We also discovered that IPNV infection induced redistribution of Myh9 to the cell membrane at the initiation of viral entry and altered the expression level of total Myh9 during viral replication. Treatments with different Myh9 inhibitors, including anti-Myh9 antibody, ML-7 and siRNA, suppressed IPNV infection. On the contrary, overexpression of Myh9 promoted IPNV infection in both susceptible and less susceptible cells. By using quantum dots-labeling technology and confocal microscopy, we tracked the entry of IPNV in real-time and found the adsorbed virions increased significantly in Myh9 overexpressed cells. Additionally, we found IPNV infection induced an increase of intracellular nanotubes and the virus could move along nanotubes to neighboring cells. Overall, our results indicate that Myh9 plays a substantial role in the entry of IPNV.