Simple SummaryNucleases are highly diverse enzymes that cleave phosphodiester bonds of nucleic acids with highly diverse enzymes. A widespread superfamily of nucleases contains PIN-like domains, which are identified in eukaryotes and prokaryotes. The domesticated silkwormBombyx mori, as a lepidoptera model insect, has numerous advantages in life science and can be used as an important model in various research areas. However, few nucleases on RNA insensitivity or virus defense have been studied inB. mori. In this study, we identified a protein asteroid (BmAst) in silkwormB. mori, containing the PIN domain and XPG domain.BmAstgene was highest expressed in hemocytes and fat body of the 5th larvae. TheBmAstgene expression was significantly induced byBombyx morinucleopolyhedrovirus (BmNPV) or dsRNA. By interference withBmAstgene expression, the proliferation of BmNPV inB. moriwas significantly increased, whereas the survival rate of silkworm larvae was significantly lower when compared with the control. Our results indicate that BmAst is involved in silkworm resistance to BmNPV infection.AbstractNuclease is a type of protein that degrades nucleic acids, which plays an important role in biological processes, including RNA interference efficiency and antiviral immunity. However, no evidence of a link between nuclease andBombyx morinucleopolyhedrovirus (BmNPV) infection in silkwormB. morihas been found. In this study, a protein asteroid (BmAst) containing the PIN domain and XPG domain was identified in silkwormB. mori.BmAstgene was highest expressed in hemocytes and fat body of the 5th instar larvae, and high expression in the pupa stage. The transcriptional levels of theBmAstgene in 5th instar larvae were significantly induced by BmNPV or dsRNA. After knocking downBmAstgene expression by specific dsRNA, the proliferation of BmNPV inB. moriwas increased significantly, whereas the survival rate of larvae was significantly lower when compared with the control. Our findings indicate that BmAst is involved in silkworm resistance to BmNPV infection.