Introduction:Cervical cancer is the third most common tumor among women. Surgery, radiotherapy, and chemotherapy are common treatments, however high stage tumors have frequently poor prognosis. Nowadays, the epigenetic reversion introduced as an efficient strategy of treatment of cervical cancer. In the process, inhibitors of DNA methyltransferase (DNMT) induce re-expression of tumor suppressor genes. Among these inhibitors, disulfiram (DSF) has been suggested as non-nucleoside analogous. In this research, we evaluated the epigenetic effect of DSF on demethylation of the tumor suppressor gene, RASSF1A, in Hela cell line. Materials and methods: Hela cells were cultured and treated with different doses from 2.5 to 37.5µM during 24, 48 and 72 hours. MTT assay was carried out to find half maximal inhibitory concentration (IC50). The methylation specific PCR (MSP) assay was applied to evaluate methylation pattern. Results: The IC50 of DSF was determined at the 2.5, 12.5, and 15µM after72 hours. The MSP results showed partial demethylation at mentioned concentrations after 72h but unmethylated band was not observed after 24h. Conclusion: Our findings indicated that, IC50 of DSF exerted a biphasic effect in Hela cell line and at least 72 hours treatment is needed for the epigenetic reversion of DSF on RASSF1Ain Hela cell line.