Mitochondria strongly contribute to the maintenance of cellular integrity through various mechanisms, including oxidative adenosine triphosphate production and calcium homeostasis regulation. Therefore, proper regulation of the abundance, distribution and activity of mitochondria is crucial for the maintenance of cellular homeostasis. Previous studies have shown that ionizing radiation (IR) alters mitochondrial functions, suggesting that mitochondria are likely to be an important target of IR. Though IR reportedly influences cellular mitochondrial abundance, the mechanism remains largely unknown. In this study, we examined how IR influences mitochondrial abundance in mouse fibroblasts. When mouse NIH/3T3 cells were exposed to X-rays, a time-dependent increase was observed in mitochondrial DNA (mtDNA) and mitochondrial mass, indicating radiation-induced upregulation of mitochondrial abundance. Meanwhile, not only did we not observe a significant change in autophagic activity after irradiation, but in addition, IR hardly influenced the expression of two mitochondrial proteins, cytochrome c oxidase subunit IV and cytochrome c, or the mRNA expression of Polg, a component of DNA polymerase gamma. We also observed that the expression of transcription factors involved in mitochondrial biogenesis was only marginally affected by IR. These data imply that radiation-induced upregulation of mitochondrial abundance is an event independent of macroautophagy and mitochondrial biogenesis. Furthermore, we found evidence that IR induced long-term cell cycle arrest and cellular senescence, indicating that these events are involved in regulating mitochondrial abundance. Considering the growing significance of mitochondria in cellular radioresponses, we believe the present study provides novel insights into understanding the effects of IR on mitochondria.