Radiation-induced cell death is thought to be caused by nuclear DNA damage that cannot be repaired. However, in this study we found that a delayed increase of mitochondrial reactive oxygen species (ROS) is responsible for some of the radiation-induced cell death in normal human fibroblast cells. We have previously reported that there is a delayed increase of mitochondrial ^·O₂^–, measured using MitoSOX™ Red reagent, due to gamma irradiation. This is dependent on Drp1 localization to mitochondria. Here, we show that knockdown of Drp1 expression reduces the level of DNA double-strand breaks (DSBs) remaining 3 days after 6 Gy irradiation. Furthermore, cells with knockdown of Drp1 expression are more resistant to gamma radiation. We then tested whether the delayed increase of ROS causes DNA damage. The antioxidant, 2-glucopyranoside ascorbic acid (AA-2G), was applied before or after irradiation to inhibit ROS production during irradiation or to inhibit delayed ROS production from mitochondria. Interestingly, 1 h after exposure, the AA-2G treatment reduced the level of DSBs remaining 3 days after 6 Gy irradiation. In addition, irradiated AA-2G-treated cells were more resistant to radiation than the untreated cells. These results indicate that delayed mitochondrial ROS production may cause some of the cell death after irradiation.