Vip3Aa is an insecticidal protein that can effectively control certain lepidopteran pests and has been used widely in biological control. However, the mechanism of action of Vip3Aa is unclear. In the present study, we showed that Vip3Aa could cause autophagy in Sf9 cells, which was confirmed by the increased numbers of GFP-Atg8 puncta, the appearance of autophagic vacuoles, and an elevated Atg8-II protein level. Moreover, we found that the AMPK-mTOR-ULK1 pathway is involved in Vip3Aa-induced autophagy, which might be associated with the destruction of ATP homeostasis in Vip3Aa-treated cells. Both the elevated p62 level and the increased numbers of GFP-RFP-Atg8 yellow fluorescent spots demonstrated that autophagy in Sf9 cells was inhibited at 24 h after Vip3Aa treatment. With the prolongation of Vip3Aa treatment time, this inhibition became more serious and led to autophagosome accumulation. Genetic knockdown of ATG5 or the use of the autophagy inhibitor 3-MA further increased the sensitivity of Sf9 cells to Vip3Aa. Overexpression of ATG5 reduced the cell mortality of Vip3Aa-treated cells. In summary, the results revealed that autophagy induced by Vip3Aa has a pro-survival role, which might be related to the development of insect resistance.