We have established “clinically relevant radioresistant (CRR) cells” that can survive exposure to 2 Gy/day X-rays for more than 30 days to overcome cancer treatment resistance. CRR cells show resistance against not only radiation but also docetaxel and hydrogen peroxide which is one of the reactive oxygen species (ROS). CRR cells produce less Fe²⁺, ROS, and lipid peroxidation compared to the parental cells. Recently, we found that this radioresistance is reversible. CRR cells that did not receive maintenance irradiation (MI; 2 Gy/day X-rays), for more than a year lost their radioresistance. We designated these CRR-NoIR cells and analyzed the mechanism of losing radioresistance. As a result, the morphology of NoIR cells maintained that of CRR cells, but the amount of both mitochondria and cytoplasm Fe²⁺ returned to the parental level. In addition, the expression of miR7-5p, which had been upregulated in CRR cells, decreased, and the expression of mitoferrin, which regulates mitochondrial iron levels downstream of miR7-5p, returned to the parental level. Furthermore, mitochondrial membrane potential and oxygen consumption also returned to parental levels. These results suggest that the amount of Fe²⁺ and the functions of mitochondria, the major iron-utilizing organelles, contribute to radio-resistance.