Obesity and type 2 diabetes are metabolic diseases, often associated with sarcopenia and muscle dysfunction. MOTS-c, a mitochondrial-derived peptide, acts as a systemic hormone and has been implicated in metabolic homeostasis. Although MOTS-c improves insulin sensitivity in skeletal muscle, whether MOTS-c impacts muscle atrophy is not known. Myostatin is a negative regulator of skeletal muscle mass and also one of the possible mediators of insulin resistance-induced skeletal muscle wasting. Interestingly, we found that plasma MOTS-c levels are inversely correlated with myostatin levels in human subjects. We further demonstrated that MOTS-c prevents palmitic acid-induced atrophy in differentiated C2C12 myotubes, whereas MOTS-c administration decreased myostatin levels in plasma in diet-induced obese mice. By elevating AKT phosphorylation, MOTS-c inhibits the activity of an upstream transcription factor for myostatin and other muscle wasting genes, FOXO1. MOTS-c increases mTORC2 and inhibits PTEN activity, which modulates AKT phosphorylation. Further upstream, MOTS-c increases CK2 activity, which leads to PTEN inhibition. These results suggest that through inhibition of myostatin, MOTS-c could be a potential therapy for insulin resistance-induced skeletal muscle atrophy as well as other muscle wasting phenotypes including sarcopenia.NEW & NOTEWORTHY MOTS-c, a mitochondrial-derived peptide reduces high-fat-diet-induced muscle atrophy signaling by reducing myostatin expression. The CK2-PTEN-mTORC2-AKT-FOXO1 pathways play key roles in MOTS-c action on myostatin expression.