Hsp90, a highly conserved cellular molecular chaperone, is involved in the life cycle of many viruses. A recent proteomics study revealed that Hsp90 was incorporated into the budded virions (BVs) of baculovirus, we therefore explored the role of Hsp90 during Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection process. The results showed that Hsp90 was essential for AcMNPV BV propagation in cultured cells. Electron microscopy detected that nucleocapsids failed to egress from the nucleus to the cytoplasm for further BV budding. Inactivation of Hsp90 abolished virus-triggered nuclear actin polymerization, a process providing essential driving forces for nucleocapsid egress. Further analyses suggested that this was due to the selectively regulation of the proper protein levels and nuclear accumulation of P40 subunit of host actin related protein 2/3 complex (Arp2/3). Thus, Hsp90 participates in baculovirus BV propagation by facilitating nuclear actin polymerization required for progeny BV production