ORF109 (Ac109) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in all sequenced baculovirus genomes, but its function is not known. This paper describes generation of an ac109 knockout virus (Ac-ac109-KO-GP) and analyses of the influence of ac109 deletion on the virus replication in Sf-9 cells so as to investigate the role of ac109 in the viral life cycle. Results revealed that budded virus (BV) yields and occlusion body synthesis were completely blocked in cells infected with the mutant virus. Electron microscopy demonstrated that ac109 deletion blocked nucleocapsid formation, though infection was initiated and electron-dense bodies associated with the virogenic stroma appeared. The mutant phenotype was rescued by an ac109 rescue virus. On the other hand, real-time PCR analysis indicated that ac109 is not required for viral DNA replication. Thus, these results suggested that ac109 plays an important role in AcMNPV nucleocapsid formation.