Ascoviruses (AVs) are insect viruses transmitted by parasitoid wasps. The unique pathology in host cells upon AV infection includes enlargement, blebbing and cleavage of host cells into virus-containing vesicles that are important in dissemination of the virus. The mechanism of pathogenesis and vesicle formation is largely unknown. Here, we explored involvement of actin filaments in virus entry, replication and pathology. The results suggested that entry of Heliothis virescens ascovirus-3e (HvAV-3e) leads to rearrangement of the actin cytoskeleton. After HvAV-3e infection, actin filaments were found in foci rather than in a homogenous distribution within the cytoplasm. Actin filaments were also found concentrating around blebs and vesiculation areas of the cell cortex following infection. Destabilization of filamentous actin by cytochalasin D did not inhibit entry or replication of the virus but affected vesiculation and pathology associated with HvAV-3e infection. These observations suggested that actin may not be required for virus entry and replication but essential for virus pathology, mainly vesicle formation.