Objective: Mild mitochondrial uncoupling has been considered as a therapeutic strategy for metabolic disorders including obesity and type 2 diabetes. Despite the beneficial effects of the mild mitochondrial uncoupling on lipid oxidation, its effects on exercise capacity has been controversial. In this study, we investigated the exercise capacity of mitochondrial targeted catalase overexpressed (MCAT) mice, which exhibited mild mitochondrial uncoupling in skeletal muscle. Methods: Mice were trained on treadmill for 4 days, and running distance was measured on 5th day and repeated for 4 weeks. During 4 weeks of exercise training, running distance of MCAT mice was decreased compared to WT mice. Results: Both mRNA. expression and protein activity of nicotinamide nucleotide transhydrogenase (NNT), an uncoupling associated gene, were increased in MCAT skeletal muscle, which could account for the decreased exercise capacity in MCAT mice. To determine whether NNT has a primary role on exercise capacity, we compared exercise capacity of C57BL6/J mice (lack of NNT gene) to C57BL6/N mice (intact NNT gene). C57BL6/N showed decreased exercise capacity compared to the J strain, and consistently supports that the reduced aerobic exercise capacity in MCAT mice is via mild mitochondrial uncoupling by increased NNT activity. Conclusion: Taken together, this study suggests that mitochondrial mild uncoupling by NNT reduces exercise capacity and has a negative impact on energy requirement in the context of increasing energy demand.