Abstract: Objective This study was designed to investingate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR)on cell growth and explore the possibility of re-expression of the hypermethylated and silenced RUNX3 gene in the hepatocarcinoma cell line HepG2. Methods The change in expression of the tumor suppressor gene RUNX3 mRNA in cultured HepG2 cells was observed by using RT-PCR before and after 5-Aza-CdR treatment. Activity of cell growth was observed by MTT assay and colony-forming test . The cell cycle was analyzed by flow cytometry . Apoptotic morphology was observed by transmitting electron microscopy. Results The gene was reactivated by two different doses of 5-Aza-CdR treatment in HepG2 cell not expressing RUNX3. The hepatocarcinoma cell line treated with 5-Aza-CdR displayed a slowed growth rate in contrast to the control group. The colony formation rate of HepG2 cell treated with 5-Aza-CdR decreased dramatically (P<0.01). The S phase was arrested and morphosis was changed as induced by 5-Aza-CdR. Conclusion 5-Aza-CdR may effectively cause demethylation and inhibit the growth of the hepatocarcinoma cell line HepG2 by reactivating the RUNX3 gene transcription silenced by aberrant hypermethylation.