Bombyx mori bidensovirus (BmBDV) is a single-stranded DNA virus belonging to the Bidensovirus genus, Bidnaviridae family. Previous studies showed that parvovirus nonstructural protein 1 (NS1) contains endonuclease, helicase, and ATPase activities and that these activities are regulated by serine/threonine phosphorylation. We have reported that residue Thr-184 site of BmBDV NS1 is phosphorylated, and that residues of Thr-181 and Thr-191 are potentially phosphorylated. However, whether phosphorylation affects BmBDV NS1 activities remains unclear. In this study, the substitution of threonine with Glycine at positions 181, 184 and 191 of BmBDV NS1 reduced its ATPase activity. After wild-type NS1 was treated with calf intestinal alkaline phosphatase (CIP), ATPase activity decreased significantly. Moreover, silkworms that were injected with recombinant viruses carrying these NS1 mutations exhibited significant increases in the median lethal time to death compared with silkworms that were injected with a virus that expressed wild-type NS1. In conclusion, these results showed that the ATPase activity and virulence of BmBDV NS1 are regulated via phosphorylation.