Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha-shl-t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up-regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F-actin. Silencing thymosin or adf or treating cells with the F-actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F-actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha-shl-t cells utilize F-actin depolymerization to suppress AcMNPV replication by up-regulating thymosin and adf. We propose Ha-shl-t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally.