Aims: Oxidative damage of DNA is very important in mutagenesis, aging and cancer. The aim of the present study was to estimate the protective effect of aqueous saffron extract on DNA damage in sulfur mustard exposed macrophage cells as a genotoxic agent.Materials & Methods: Macrophage cells were obtained from mice peritoneum. The viability of cells was determined and then they were treated with KMnO4, SM and SM+saffron extract in culture media. The SM toxicity in macrophage cells was determined using MDA and GSH concentrations (nM/mg protein) and the extent of genotoxicity was investigated using alkaline comet assay and reported as the movement of DNA in electrophoretic field and %DNA remaining in the nucleus.Results: The concentration of MDA and GSH in normal cells and KMnO4, SM and SM+saffron extract treated cells were respectively 1.9±0.18, 9.4±0.95, 9.3±1.2, 5.1±0.55 and 9.6±0.71, 2.4±0.32, 2.5±0.21, 4.81±0.39 nM/mg protein. The comet result showed that DNA movement in normal cell was 23.1±1.43mm, but in KMnO4, SM and SM+saffron extract treated cells were respectively 39.75±4.92, 40.45±4.42 and 28.33±4.6mm.Similarly, the percent of DNA in nucleus in normal, KMnO4, SM and SM+saffron extract treated cells were respectively %99.3±3.6, 20.7±3.8, 22.61±4.3 and 65.5±4.8 that showed the significant protective effect of saffron extract on reduction of DNA damage in SM contaminated cells.Conclusion: The results demonstrate the anti-genotoxic effect of saffron aqueous extract against the oxidative damage of DNA caused by SM. However, the genotoxic activity of SM significantly reduced by treatment with aqueous saffron extracts. Therefore, it seems that the oral supplement of saffron aqueous extract can probably prevent the complications of these oxidative agents.