Exposure of fluorine at toxic concentrations causes serious damage by accumulating in especially bones, kidneys, and other soft tissues. Fluorine at cytotoxic concentrations may cause DNA damage. This study aims to determine the level of DNA damage due to sodium fluoride (NaF) at different hours (3rd, 12th, and 24th hours) and in IC(50)concentrations designated for each hour and reveal the protective effect of lycopene on possible damage. The best enhancer concentrations (1 mu M) of microtitration (MTT) viability test and proliferation of lycopene and IC(50)values of NaF at the 3rd, 12th, and 24th hour were 9600, 5500, and 3200 mu M, respectively. DNA damage significantly increased in all NaF-treated groups in comparison with the control group (p < 0.05). DNA damage due to NaF+LYC application significantly decreased in comparison with the control group (p < 0.05). Lycopene application significantly increased the expression levels of the Ku70 and Ku80 genes which have a part in DNA repair (p < 0.05). The statistical data showed that application of lycopene which is an important antioxidant molecule may be beneficial for decreasing NaF-induced DNA damage. In conclusion, applying lycopene for cytotoxicity due to fluorine in NRK-52E cell line had different effects based on the dosage and time; thus, it can be a potential option for preventing fluorosis-induced toxicity and developing new treatment approaches.