Nucleopolyhedrovirus (NPV) can alter its host behaviour such that infected larvae hang at the top of trees before their death. This phenomenon was firstly described by Hofmann in 1891 and named as "tree-top disease". Subsequent studies have described effects during the infection proceedings as NPVs manipulate the host to avoid the immune response, cross defensive barriers and regulate hormones. In this study, we demonstrate that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway is involved in host manipulation by Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Particularly at the late stage of infection, a multifunctional dephosphorylase in the PI3K/AKT signaling pathway is dynamically upregulated, namely, the phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase ( PTEN ) gene. The biological assays of PTEN gene knockdown showed that an increase in PTEN gene expression was necessary for the infected Lymantria dispar larvae's terminal climbing behavior, death postponement and virion production. The results imply that the PI3K/AKT signaling pathway and PTEN gene might play an essential role in "tree-top disease" induced by LdMNPV.