MOTS-c is an exercise mimetic and improves insulin sensitivity in aged and diet-induced obese mice. Although plasma markers are good markers for the metabolic condition, whether MOTS-c changes plasma markers in diet-induced obese mice has not been examined. Here, we used an unbiased metabolomics approach to examine the effect of MOTS-c on plasma markers of metabolic dysfunction. We found that three pathways - sphingolipid metabolism, monoacylglycerol metabolism, and dicarboxylate metabolism - were reduced in MOTS-c-injected mice. Interestingly, these pathways are upregulated in obese and T2D models. MOTS-c improves insulin sensitivity and increases beta-oxidation to prevent fat accumulation in DIO mice through these pathways. These results provide us a better understanding of the mechanism of how MOTS-c improves insulin sensitivity and reduces the body weight and fatty liver and opens a new venue for further study.,© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.