Background: Melatonin has been suggested to have antiproliferative effects on cancer cells. These effects can be attributed to immunomodulation, growth factor inhibition, induction of apoptosis and prooxidant properties. Melatonin is considered as a safe drug with minimal adverse effects. Objectives: We planned to investigate the effects of melatonin in hepatoma (Hep G2) cell line. In this study, different concentrations of melatonin were studied to assess its effects on human hepatoma (Hep G2) cell line in vitro. Methods: In this study, different doses (5×10-5M, 5×10-4M, 10-3M) of melatonin were administered into hepa-tocellular carcinoma cell line in vitro. After an incubation period of 72 hours, the studied and control groups were evaluated for cell cycle, morphology, proliferating index and apoptosis percentage. Results: A significant decrease in percentage of phase G0/G1 cells was found in high-dose melatonin group (10-3M) compared to control group. Melatonin increased the cell counts in S phase of cell cycle at high doses as well. However, phase G2/M cell percentage did not change with the administration of melatonin. Cell proliferation was increased in all melatonin groups, but the only statistically significant difference was found between the high-dose and control groups. There was a significant increase in proliferative index between the control group and high-dose melatonin group. Conclusion: High dose of melatonin increases the cell count in S phase and shows an antiproliferative effect on hepatoma cells. This indicates that melatonin can be considered a promising drug when used along with other antineoplastic agents for the treatment of hepatoma.However, it has no effect on apoptosis and colony counts.