Consumption of a high fat diet (HFD) and being overweight both induce functional deterioration and atrophy of the hippocampus. These alterations are associated with mental disorders such as depression and anxiety. Exercise combats obesity and enhances brain health. There is substantial evidence that neuronal nitric oxide synthase (nNOS) is a key regulator of affective behavior, and that increased brain nNOS leads to anxiety while environmental enrichment (EE), which reduces brain nNOS, has anxiolytic effects. In this study we investigated the effects of HFD with and without exercise on nNOS protein and gene expression levels in the brains of mice. Twelve weeks of HFD consumption increased body and mesenteric fat weight, as well as nNOS protein levels in the hippocampus and cerebral cortex. Six weeks of exercise training reduced body fat and rescued hippocampal and cortical nNOS expression levels in HFD-fed mice. Cerebellar nNOS expression was unaffected by HFD and exercise. Our results suggest that HFD-induced brain dysfunction may be regulated by hippocampal and/or cortical nNOS, and that exercise may have therapeutic potential for the treatment of HFD-induced depression and anxiety via the nNOS/NO pathway. In conclusion, exercise reverses HFD-induced changes in hippocampal and cortical nNOS protein levels in mice. (C) 2017 Elsevier Inc. All rights reserved.