The SNF2 global transactivator gene homologue (Bm33) of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the genes exclusive to group I NPVs, but its function remains unknown. This study describes the characterization of Bm33. Transcriptional analysis suggested that Bm33 is an early gene, as its transcript was observed at 4 h post-infection in BmNPV-infected BmN cells. To examine the role of Bm33 during BmNPV infection, a Bm33 deletion mutant (BmORF33D) was constructed and its infectivity was characterized in BmN cells and B. mori larvae. BmORF33D did not have any obvious defects in the production of budded viruses (BVs) or occlusion bodies (OBs) in BmN cells compared with wild-type BmNPV. Larval bioassays revealed that deletion of Bm33 did not reduce virus infectivity. However, BmORF33D took approximately 10–15 h longer than wild-type BmNPV to kill B. mori larvae when tested by either BV injection or OB ingestion. These results suggest that Bm33 is not essential for virus growth in vitro or in vivo, but that it accelerates the time of death of B. mori larvae.