OBJECTIVES: To determine the effect of 5-aza-2'-deoxycytidine (DAC) on human endometrial carcinoma cell (HECC) oncogenicity and demonstrate a molecular mechanism by which DAC modulates HECC oncogenicity. METHODS: The effect of DAC was tested on HECC RL95-2, AN3, Ishikawa and ECC1 cells. The role of 14-3-3σ on HECC oncogenicity in response to DAC treatment was evaluated in RL95-2 and AN3 cells after forced expression or silencing of 14-3-3σ gene expression. RESULTS: Treatment of HECC with DAC produced non-cytotoxic cell growth inhibition and G2/M cell cycle arrest. This effect was strongly correlated with increased expression of p21 and 14-3-3σ. Silencing of 14-3-3σ induced cellular proliferation and reduced the effect of DAC on cell cycle arrest in G2/M phases. Conversely, forced expression of 14-3-3σ showed the opposite effect. Furthermore, forced expression of 14-3-3σ in human endometrial cell lines reduced cell growth and colony formation. CONCLUSIONS: We suggest that 14-3-3σ in HECC suppresses cell proliferation and mediates DAC induced G2/M arrest and inhibition of cell proliferation in HECC.