After mitochondria colonized eukaryotic cells more than a billion years ago, they acquired numerous functions over the course of evolution, such as those involved in controlling apoptosis, autophagy, and cellular metabolism together with host cells. The major tumor suppressors, p53 and ARF in the nucleus also participate in such crosstalk between host cells and mitochondria by activating p53 target genes involved in varied mitochondrial functions. However, recent evidence suggests that p53 and ARF can also directly localize to mitochondria and contribute to this cross talk to maintain tissue homeostasis for the prevention of various diseases. Here, we discuss the functions of mitochondrial p53 and ARF via interactions with mitochondrial proteins as well as the mechanism of the localization of p53 and ARF to mitochondria. Because mitochondrial dysregulation is involved in the development of several disease types, such as cancer, neurodegenerative diseases, and age-related diseases, understanding the roles of p53 and ARF in mitochondria may facilitate the development of novel mitochondrial-specific drug targets against such diseases.