orf74 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homologue of uncharacterized orf91 of Autographa californica multicapsid NPV, predicted to encode a protein of 17.3 kDa. RT-PCR showed that the transcription of orf74 was initiated at 12 h post-infection stage. Expression assay indicated that ORF74 localized in the nucleus of infected BmN cells. To study the function of ORF74, an orf74-knockout virus was constructed using bacmid technology. Analysis of the production of budded virions and occlusion bodies, the formation of nucleocapsids, and the level of viral DNA revealed no significant difference among the mutant, the control, and the orf74-repaired virus. Interestingly, bioassay showed that the median lethal time of the orf74-knockout virus was 14.7 h longer than the control virus, but the virus yield was similar to that of control virus. Thus, our data indicated that ORF74 is most likely implicated in the virulence of BmNPV, and is not essential for virus replication.