Endometrial cancer (EC) is one of the most common gynecologic malignancies. However, the molecular mechanisms underlying the development and progression of EC remain unclear. Here, we demonstrated that the protein proviral insertion in murine lymphomas 2 (PIM2) was necessary for maintaining EC tumorigenesis in vivo and in vitro, and could inhibit AMPKα1 kinase activity in EC cells. Specifically, we found that PIM2 bound to AMPKα1, and directly phosphorylated it on Thr467. Phosphorylation of AMPKα1 by PIM2 led to decreasing AMPKα1 kinase activity, which in turn promoted aerobic glycolysis and tumor growth. In addition, PIM2 expression positively correlated with AMPKα1 Thr467 phosphorylation in EC tissues. Further, treatment with a combination of the PIM2 inhibitor SMI-4a and the AMPKα1 activator AICAR could effectively inhibit tumor growth. Thus, our findings provide insight into the role of PIM2 and AMPKα1 in EC and suggest that combination targeting of these proteins may represent a new strategy for EC treatment.