Among mitochondrial NADP-reducing enzymes, nicotinamide nucleotide transhydrogenase (NNT) establishes an elevated matrix NADPH/NADP by catalyzing the reduction of NADP at the expense of NADH oxidation coupled to inward proton translocation across the inner mitochondrial membrane. Here, we characterize NNT activity and mitochondrial redox balance in the brain using a congenic mouse model carrying the mutated Nnt gene from the C57BL/6J strain. The absence of NNT activity resulted in lower total NADPH sources activity in the brain mitochondria of young mice, an effect that was partially compensated in aged mice. Nonsynaptic mitochondria showed higher NNT activity than synaptic mitochondria. In the absence of NNT, an increased release of H O from mitochondria was observed when the metabolism of respiratory substrates occurred with restricted flux through relevant mitochondrial NADPH sources or when respiratory complex I was inhibited. In accordance, mitochondria from Nnt brains were unable to sustain NADP in its reduced state when energized in the absence of carbon substrates, an effect aggravated after H O bolus metabolism. These data indicate that the lack of NNT in brain mitochondria impairs peroxide detoxification, but peroxide detoxification can be partially counterbalanced by concurrent NADPH sources depending on substrate availability. Notably, only brain mitochondria from Nnt mice chronically fed a high-fat diet exhibited lower activity of the redox-sensitive aconitase, suggesting that brain mitochondrial redox balance requires NNT under the metabolic stress of a high-fat diet. Overall, the role of NNT in the brain mitochondria redox balance especially comes into play under mitochondrial respiratory defects or high-fat diet.