The increasing incidence rate and decreasing patients’ five-year survival rate for endometrial cancer (EC) in recent decades highlight the necessity for further investigation of the molecular characteristics involved in cancer initiation and progression. In this study, we found that the pathways associated with mitotic cell cycle were enriched in primary EC samples versus normal endometrial samples through analyzing RNA-seq data of The Cancer Genome Atlas (TCGA). The messenger RNA (mRNA) expression of three activator E2Fs (E2F1, E2F2, and E2F3) and their target genes increased significantly in EC samples. Additionally, the high transcriptional activity of activator E2Fs was associated with poor survival, advanced clinical stage, high histologic grade, and aggressive histological type. We further demonstrated that E2Fs hyperactivation correlated with DNA hypomethylation and high cyclin-dependent kinase 4 (CDK4) expression. Moreover, abemaciclib, a selective CDK4 inhibitor, significantly inhibited the proliferation rates of human EC cell lines in vitro. And, abemaciclib also obviously inhibited EC cell growth in nude mice model. Collectively, our data suggest that the misregulation of CDK4/RB/E2Fs axis is associated with EC oncogenesis, and abemaciclib is a potential targeted drug for EC.