Bombyx mori Tryptophanyl-tRNA synthetase (BmWARS) belongs to the family of Ic-like aminoacyl-tRNA synthetases (aaRSs), whose specific recognition of the substrate Trp, tRNA, maintains the fidelity of protein synthesis. In this study, BmWARS was cloned and characterized from the midgut of the silkworm, Bombyx mori, resulting in an open reading frame (ORF) with a full length of 1,149 bp, which can encode 382 Aa. BmWARS is localized in the cytoplasm, and is expressed in all tissues of the silkworm, with higher expression in the testis, ovary, silk gland and malpighian tubule. The expression of BmWARS was significantly up-regulated in the midgut and silk gland after infection with Bombyx mori nuclear polyhedrosis virus (BmNPV). In addition, overexpression of BmWARS inhibited BmNPV infection and replication extremely significantly, while interference with BmWARS expression promoted BmNPV infection and replication. Analysis of the immune pathways in which BmWARS may be involved revealed that the expression of the key genes of the PI3K-Akt pathway, BmPI3K, BmAkt, BmPDK1, BmeIF4E, BmS6, and p-Akt protein was significantly reduced, whereas the expression of BmPTEN, BmFoxO, and BmCaspase9 was significantly increased in the cells that overexpressed BmWARS and were infected with BmNPV. Meanwhile, the results of the study interfering with the expression of BmWARS were completely opposite to those of the study overexpressing BmWARS. This is the first report that BmWARS has antiviral effects in Bombyx mori. Moreover, BmWARS inhibits BmNPV infection and replication in Bombyx mori cells by promoting apoptosis and inhibiting cell proliferation.