Background and Aims: A novel bioactive peptide, mitochondrial-derived peptide (MOTS-c), has recently attracted attention as a potential prevention or therapeutic option for obesity and type 2 diabetes mellitus (T2DM). MOTS-c profiles have not yet been reported in human obesity and T2DM. We aimed to determine circulating MOTS-c levels in obesity and explore the association between MOTS-c levels and various metabolic parameters. Methods: In this case-control study, 40 obese children and adolescents (27 males) and 57 controls (40 males) were recruited in the Hubei Province of China in 2017. Circulating MOTS-c levels were measured, clinical data (eg, glucose, insulin, and lipid profile) were recorded, and anthropometric measurements were performed. Finally, we investigated correlations between MOTS-c levels and related variables. Results: MOTS-c levels were significantly decreased in the obese group compared with the control group (472.61 +/- 22.83 vs 561.64 +/- 19.19 ng/mL, P < .01). After classification by sex, MOTSc levels were significantly decreased in obese male children and adolescents compared to their counterparts (465.26 +24.53 vs 584.07 +21.18 ng/mL, P < .001), while they were comparable between the obese and healthy female subjects (487.89 +/- 49.77 vs 508.85 +/- 38.76 ng/mL, P > .05). Further, MOTS-c levels were negatively correlated with body mass index (BMI), BMI SD score, waist circumference, waist-to-hip ratio, fasting insulin level, homeostasis model assessment of insulin resistance (HOMA-IR), and glycated hemoglobin (HbA1c) in the male cohort. Conclusions: Circulating MOTS-c levels were decreased in obese male children and adolescents and correlated with markers of insulin resistance and obesity.