Background: Melatonin is a natural antioxidant that is produced by the pineal gland. In this study was evaluated antioxidant and possible protective effects of melatonin on frequency of micronucleus (MN) formation in human cell lines exposed to γ-radiation. Materials and Methods: To achieve the best concentration for antioxidant activity of melatonin DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used. Hela and MRC5 cells were cultured and treated with optimum concentration of melatonin (1200μg/ml). After 2h, cells were exposed to 2Gy-gamma ray. For each cell group, one flask was considered as control. Immediately after irradiation, cells were exposed to cytochalasin B to arrest cells at cytokinesis. Then the frequency of MN induced by radiation alone or in the presence of melatonin was evaluated.  Results: By DPPH assay, the optimum concentration of melatonin for its antioxidant activity was determined 1200μg/ml. Our results showed that the frequency of micronuclei increased in irradiated cells compared to the control groups (p<0.05). Conversely, pre-treatment of cells with melatonin significantly reduced the number of MN produced both in MRC5 and Hela cells (p<0.05). Conclusion: Results indicated that γ-radiation induced MN in the cells. The protective effect was achieved when melatonin was present in the cellular environment pre-irradiation. Indeed, melatonin with scavenging and antioxidant ability neutralizes toxic reactants and stimulates DNA repair pathways. Moreover, the results indicate that protective effect of melatonin is higher in MRC5 cells than in Hela cells. Therefore, it can be concluded that other mechanisms such as induction of cell cycle arrest by melatonin might be exist only in MRC5 cells. However, the radio-protective mechanism of melatonin is not clearly known.