We previously showed that rRNA cleavage and degradation occur in Bombyx mori BM-N cells upon infection with heterologous NPVs, including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV, and Spodoptera litura MNPV. Additionally, transient expression assay analyses suggested that viral P143s, which are baculovirus DNA helicases that are essential for viral DNA replication, are responsible for the observed rRNA cleavage and degradation. Here, we examined whether viral DNA replication is related to rRNA cleavage and degradation in AcMNPV-infected BM-N cells using an AcMNPV temperature-sensitive-mutant (ts8) defective in P143 function at non-permissive temperature. Electrophoretic analyses of total RNAs from ts8-infected BM-N cells revealed that rRNA cleavage and degradation still occurred in the absence of viral DNA replication at non-permissive temperature. In addition, transfection analysis with an ac-p143 null AcMNPV bacmid demonstrated that the p143 gene is responsible for rRNA cleavage and degradation in AcMNPV-infected BM-N cells. Taken together, these results indicate the possibility that the viral DNA replication-related function of P143 is not associated with rRNA cleavage and degradation in NPV-infected BM-N cells.