Homologs of Autographa californica nucleopolyhedrovirus ORF43 (Ac43) are found in all group I and most group II NPVs, but their functions remain unknown. In Bombyx mori NPV (BmNPV)-infected BmN cells, Bm34, a BmNPV homolog of Ac43, is expressed as a late gene and its product is localized in the nucleus. To examine the role of Bm34 during BmNPV infection, we constructed a Bm34 deletion mutant (BmORF34D) and characterized its infectivity in BmN cells and B. mori larvae. BmORF34D produced far fewer occlusion bodies (OBs) in BmN cells as compared with wild-type BmNPV. This reduction is assumed to be mainly due to the transcriptional down-regulation of two genes, very late expression factor (vlf-1) and few polyhedra (fp25K), both of which are required for efficient polyhedrin expression. Larval bioassays also revealed that Bm34 accelerates death of B. mori larvae. These results suggest that Bm34 is required for efficient late and very late gene expression.