Insulin resistance (IR) increases the risk for Alzheimer’s disease and other dementias; however, the underpinning mechanisms for this increased risk remain to be fully defined. Impaired mitochondrial function is one component contributing to cognitive impairment. PURPOSE: As insulin resistance impairs mitochondrial oxidative metabolism and increases reactive oxygen species (ROS) in skeletal muscle, we considered whether similar events occur in the brain, which like muscle is rich in insulin receptors and mitochondria. Further, we sought to determine whether aerobic exercise (AE) could prevent the hypothesized deficits in mitochondrial function accompanying diet-induced IR. METHODS: 12-week-old, male, C57BL6 mice were fed a standard (Chow) or high fat diet (HFD) (60% kcal from fat) for four-weeks and provided access to running wheels (EX) or sedentary (SED) conditions (n = 9-10 per group). Following treatment, mitochondria were freshly isolated from the cerebrum to assess mitochondrial respiration, ROS production, and ATP production. Insulin resistance was determined ex-vivo in the hippocampus by the ability of insulin to stimulate AKT-phosphorylation. mtDNA copy number, mRNA expression, and proteomic measurements were performed on isolated hippocampal tissue. RESULTS: HFD induced hippocampal insulin resistance (p < 0.001), which was corrected by AE. HFD decreased ATP production 12% (p = 0.01) and increased ROS emission 79% (p < 0.01) in isolated cerebral mitochondria, which were rescued with AE. Impairments in mitochondrial function with HFD were paralleled by reductions in mtDNA copy number (1.00 ± 0.06 vs 0.85 ± 0.06; p = 0.02) and mRNA expression of mitochondrial genes, such as PGC1a1 (1.00 ± 0.05 vs 0.78 ± 0.07; p = 0.03) and TFAM (1.00 ± 0.08 vs 0.62 ± 0.11; p = 0.03), which were corrected by AE. Proteomic analysis of the hippocampus showed that HFD led to oxidative post-translational modifications (PTMs) to 17 mitochondrial proteins (corrected p-value ≤ 0.05 and absolute log2 fold change ≥ 0.5); however, this increase in oxidative PTMs to mitochondrial proteins with HFD was almost completely reversed by AE. CONCLUSIONS: HFD induces IR in the cerebrum and hippocampus, which associates with mitochondrial dysfunction. Brain IR and mitochondrial dysfunction accompanying HFD are prevented with AE.