Uterine serous carcinoma (USC) is a subtype of endometrial cancer. Compared with endometrial endometroid carcinoma, the majority of USC cases are more aggressive. Cyclin-dependent kinase inhibitor 2A (P16INK4A) is a canonical tumor suppressor that blocks cell cycle progression; however, P16INK4A is overexpressed in USC. The aim of the present study was to determine the role of P16INK4A in P16INK4A‑positive endometrial cancer, with the hope of elucidating a novel therapeutic approach for this type of malignancy. A total of 2 endometrial cancer cell lines, ETN‑1 and EFE‑184, were selected for further investigation, due to them being known to express high levels of P16INK4A. Using short hairpin RNA targeting P16INK4A, P16INK4A was downregulated in these cancer cell lines. Cell viability and migration were examined via 2D/3D clonogenic and wound healing assays. Subsequently, GSK‑J4, a histone demethylase inhibitor, was employed to deplete P16INK4A in these cancer cell lines and an ex vivo culture system of a patient‑derived xenograft (PDX) endometrial tumor sample. Following P16INK4A knockdown, the proliferation and migration of ETN‑1 and EFE‑184 cells markedly declined. When exposed to GSK‑J4, the levels of KDM6B and P16INK4A were almost completely abrogated, and the cell viability was significantly reduced in these cell lines and the ex vivo‑cultured PDX tumor explants. The association between the levels of P16INK4A, lysine demethylase 6B (KDM6B) and the methylation status of histone 3 lysine 27 (H3K27) in these cell lines and the human USC tumor sample was also demonstrated. P16INK4A appears to be oncogenic in a number of endometrial cancer cell lines. The level of P16INK4A is associated with the methylation status of H3K27. Increased methylation of H3K27 coexists with downregulation of KDM6B and, subsequently, P16INK4A, which reduces cell proliferation and invasiveness in endometrial cancer. The observations of the present study may enable the development of a novel therapeutic strategy for P16INK4A‑positive endometrial cancer, particularly USC.