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In <i>Bombyx mori</i>, as an important economic insect, it was first found that some strains were completely refractory to infection with <i>Autographa californica</i> nucleopolyhedrovirus (AcMNPV) through intrahemocelical injection; whereas almost all natural strains had difficulty resisting <i>Bombyx mori</i> nucleopolyhedrovirus (BmNPV), which is also a member of the family Baculoviridae. Previous genetics analysis research found that this trait was controlled by a potentially corresponding locus on chromosome 3, but the specific gene and mechanism was still unknown. With the help of the massive silkworm strain re-sequencing dataset, we performed the Genome-Wide Association Studies (GWAS) to identify the gene related to the resistance of AcMNPV in this study. The GWAS results showed that the Niemann-Pick type C1 (NPC-1) gene was the most associated with the trait. The knockdown experiments in BmN cells showed that <i>BmNPC1</i> has a successful virus suppression infection ability. We found a small number of amino acid mutations among different resistant silkworms, which indicates that these mutations contributed to the resistance of AcMNPV. Furthermore, inhibition of the <i>BmNPC1</i> gene also changed the viral gene expression of the AcMNPV, which is similar to the expression profile in the transcriptome data of p50 and C108 strains.