Objective: Given the importance of mitochondria function in fatty acid oxidation (FAO) and insulin resistance, changes in mitochondrial NAD(P)H pool could affect tissue lipid accumulation and metabolic diseases. Catalase has NAD(P)H binding ability and mitochondrial-targeted catalase overexpressed (MCAT) mice has been shown a protection from diet-induced insulin resistance. However, only redox association is reported and other detailed mechanisms linking catalase to insulin signaling (eg. Tissue lipid, mitochondrial function) has not yet been fully understood. Methods: To test whether mitochondrial FAO is a mechanism accounted for the protection of MCAT mice, we determine mitochondrial respiratory function, energy balance, insulin sensitivity and intramuscular lipid contents in MCAT and littermate control (WT) mice on high-fat diet (HFD). Results: After HFD feeding, MCAT mice are protected from muscle insulin resistance, which is accounted by decreased muscle lipid accumulation and increased energy expenditure. The changes in lipid and energy metabolism are associated with increased nicotinamide nucleotide transhydrogenase (NNT) expression and activity, which use mitochondrial proton gradient to produce NADPH. Accordingly, mitochondrial FAO is increased in both muscle and mouse embryonic fibroblast (MEF) cells from MCAT mice without increasing ATP contents, indicating NNT-mediated mitochondrial uncoupling. Furthermore, chemical NNT inhibitor dramatically reduces mitochondrial fatty acid oxidation in MEF cells. 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.