Simple SummaryEcdysone is an essential factor affecting host immunity, in addition to regulating insect metamorphosis. There are two isoforms of ecdysone receptors (EcR) in silkworm, BmEcR-B1, and BmEcR-A. In this study,BmEcR-B1was found to be more critical than BmEcR-A in responding to BmNPV infection. Further studies confirmed thatBmEcR-B1played an anti-BmNPV role in the participation of 20E, which was associated with 20E-induced apoptosis. This study provides theoretical support for elucidating the resistant mechanism of silkworm to BmNPV and the control of BmNPV disease in sericulture.AbstractBombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation betweenBombyx mori ecdysone receptor B1(BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics ofBmEcR-B1and its isoform,BmEcR-A, were initially analyzed.BmEcR-B1was found to be more critical thanBmEcR-Ain silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showedBmEcR-B1had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore,BmEcR-B1was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection.