The impact of the mitochondria‐targeted antioxidant MitoQ was evaluated in the metabolic alterations and the adipose tissue remodeling associated with obesity. Male Wistar rats were fed either a high‐fat diet (HFD; 35% fat) or a standard diet (3.5% fat) for 7 wk and treated with MitoQ (200 μM). A proteomic analysis of visceral adipose tissue from patients with obesity and patients without obesity was performed. MitoQ partially prevented the increase in body weight, adiposity, homeostasis model assessment index, and adipose tissue remodeling in HFD rats. It also ameliorated protein level changes of factors involved in insulin signaling observed in adipose tissue of obese rats: reductions in adiponectin and glucose transporter 4 (GLUT 4) and increases in dipeptidylpeptidase 4, suppressor of cytokine signaling 3 (SOCS3), and insulin receptor substrate 1 phosphorylation. MitoQ prevented down‐regulation of adiponectin and GLUT 4 and increases in SOCS3 levels in a TNF‐α‐induced insulin‐resistant 3T3‐L1 adipocyte model. MitoQ also ameliorated alterations in mitochondrial proteins observed in obese rats: increases in cyclophylin F and carnitine palmitoyl transferase 1A and reductions in mitofusin1, peroxiredoxin 4, and fumarate hydratase. The proteomic analysis of the visceral adipose tissue from patients with obesity show alterations in mitochondrial proteins similar to those observed in obese rats. Therefore, the data show the beneficial effect of MitoQ in the metabolic dysfunction induced by obesity.—Marín‐Royo, G., Rodríguez, C., Le Pape, A., Jurado‐Lopez, R., Luaces, M., Antequera, A., Martinez‐Gonzalez, J., Souza‐Neto, F. V., Nieto, M. L., Martínez‐Martínez, E., Cachofeiro, V. The role of mitochondrial oxidative stress in the metabolic alterations in diet‐induced obesity in rats. FASEB J. 33, 12060‐12072 (2019). http://www.fasebj.org