Several studies have demonstrated that mitochondria are critically involved in the pleiotropic manifestation of radiation effects. While conventional whole-cell irradiation compromises the function of mitochondria, the effects of subcellular targeted radiation are not yet fully understood. In this study, normal human diploid cells with cell-cycle indicators were irradiated using a synchrotron X-ray microbeam, and mitochondrial membrane potential was quantified by JC-1 over the 72-h period postirradiation. Cytoplasmic irradiation was observed to temporarily enlarge the mitochondrial area with high membrane potential, while the total mitochondrial area did not change significantly. Unexpectedly, cell-nucleus irradiation promoted a similar increase not only in the mitochondrial areas with high membrane potential, but also in those with low membrane potential, which gave rise to the apparent increase in the total mitochondrial area. Augmentation of the mitochondrial area with low membrane potential was predominantly observed among G₁ cells, suggesting that nucleus irradiation during the G₁ phase regulated the mitochondrial dynamics of the cytoplasm, presumably through DNA damage in the nucleus.