Mitochondrial biogenesis and mitophagy are recognized as critical processes underlying mitochondrial homeostasis. However, the molecular pathway(s) coordinating the balance between these cellular programs is still poorly investigated. Here, we show an induction of the nuclear and mitochondrial peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) during myogenesis, which in turn co-activates the transcription of nuclear and mtDNA-encoded mitochondrial genes. We demonstrate that PGC-1α also buffers oxidative stress occurring during differentiation by promoting the expression of antioxidant enzymes. Indeed, by downregulating PGC-1α, we observed an impairment of antioxidants expression, which was accompanied by a significant reactive oxygen species (ROS) burst and increase of oxidative damage to proteins. In parallel, we detected a decrease of mitochondrial mass and function as well as increased mitophagy through the ROS/FOXO1 pathway. Upon PGC-1α downregulation, we found ROS-dependent nuclear translocation of FOXO1 and transcription of its downstream targets including mitophagic genes such as LC3 and PINK1. Such events were significantly reverted after treatment with the antioxidant Trolox, suggesting that PGC-1α assures mitochondrial integrity by indirectly buffering ROS. Finally, the lack of PGC-1α gave rise to a decrease in MYOG and a strong induction of atrophy-related ubiquitin ligases FBXO32 (FBXO32), indicative of a degenerative process. Overall, our results reveal that in myotubes, PGC-1α takes center place in mitochondrial homeostasis during differentiation because of its ability to avoid ROS-mediated removal of mitochondria.