Objective Investigate the effect of Zebularine on methylation state and expression of RASSF1A gene in human ovarian cancer cell line A2780, and to discuss the mechanism of RASSF1A gene silencing in human ovarian cancer cell as well as the regulating effect of demethylation agent on its expression . Methods A2780 cells were cultured in RPMI1640 and were treated with different concentrations (50μmol/L, 100μmol/L, 200μmol/L) of DNA methyltransferase inhibitor Zebularine. Methylation-specific PCP(MSP) was used to detect promoter methylation state of RASSF1A gene. RT-PCR and Western blot were used to detect expression of mRNA and protein of RASSF1A gene before and after treatment with Zebularine respectively. Results Promoter hypermethylation of RASSF1A gene was detected in ovarian cancer cell A2780, and no expression was detected before treatment. After treated with Zebularine,the promoter region of RASSF1A gene exhibit demethylation state ,and its expression occured at mRNA and protein level.Conclusion Promoter hypermethylation is the mechanism of RASSF1A gene silencing in human ovarian cancer cell line, and Zebularine can reverse RASSF1A gene’methylation state and regulate its expression.