VP39 is the major capsid protein of Heliothis armigera nucleopolyhedrovirus (HaSNPV), and it might have induced the aggregation of host cellular actin in vitro in our previous study. We demonstrated here that VP39 could interact with host actin in vivo in Helicoverpazea (Hz-AM1 cells) through coimmunoprecipitation assay. With confocal immunofluorescence microscopy, it was confirmed further that the released HaSNPV nucleocapsids/VP39s in the host cytoplasm (0.5 hours after infection) colocalized where the actin aggregated and that the nucleocapsids/VP39s were transported from the host cytoplasm to the nucleus (2 hours after infection). Because cytochalasin D (CD) was used to prevent host global actin from forming filamentous structures, the infection efficiency of the recombinant virus HaSNPV/gfp Delta p74, with the gfp gene inserted into HaSNPV p74 gene loci, was decreased to 7.34%, whereas it was 34.7% in normal host cells and 55.7% in the cells whose microtubules had been destroyed by colchicin. Ultramicroscopy assay revealed that HaSNPV nucleocapsids could enter the cytoplasm of CD-treated cells but could not be transported to the nucleus, which resulted in the lower infection efficiency of HaSNPV/gfp Delta p74 in CD-treated cells. However, transportation of the nucleocapsids was not inhibited in colchicin-treated cells, demonstrating that the transportation of HaSNPV nucleocapsid from the cytoplasm to the nucleus was associated with actin filaments but not with microtubules, a conclusion that is also strongly supported by evidence from the RNAi interference of host actin during HaSNPV infection.