ORF69 (Ac69) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in Spodoptera frugiperda Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an ac69 knockout AcMNPV bacmid mutant and analyses of the influence of ac69 deletion on the viral infectivity in Sf-9 cells and Trichoplusia ni larvae so as to investigate the role of ac69 in the viral life cycle. Results indicated that ac69 deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect AcMNPV infectivity for Trichoplusia ni larvae in LD50 and LT50 bioassay when administered orally. These results suggest that ac69 may be dispensable for viral infectivity both in vitro and in vivo.