Late expression factor 11 (LEF-11) is an essential protein in the regulation of Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication and late gene expression. Our recent quantitative analysis of protein acetylome revealed for the first time that LEF-11 can be acetylated at one lysine residue (K83) during viral infection, but the underlying mechanism is unclear. The acetylation level for K83 was down-regulated after 36 h post-infection by approximately 30%. To clarify the regulatory function of this modification, overlap PCR was used for site-specific mutagenesis for acetylated (K83Q) or deacetylated (K83R) mimic mutants of LEF-11. The results of viral titration and quantitative polymerase chain reaction showed that after K83 acetylation, budding virion production and the viral genome replication level were significantly upregulated. Meanwhile, the results of yeast two-hybrid (y2H) system confirmed that K83 deacetylation modification inhibited the interaction between LEF-11 and immediate early gene 1 (IE-1). In conclusion, the acetylation of LEF-11 at K83 might enhance the interaction with IE-1 in the host cell nucleus to promote viral DNA replication, and might be one of the antiviral strategies of the silkworm host. The host inhibits virus proliferation by deacetylating LEF-11.