The central nervous system (CNS) integrates peripheral hormonal signals to regulate glucose homeostasis and feeding behavior. Obesity can cause the development of insulin resistance in the brain and completely disrupt the regulative functions of the CNS. In rodents, the Dorsal Vagal Complex (DVC) of the brain senses insulin to regulate glucose metabolism and 3 days high fat diet feeding (HFD) completely disrupts the insulin response. Mitochondria undergo morphological and metabolic changes to maintain energy homeostasis in eukaryotic cells. Whether specific changes of mitochondria dynamics directly alter cellular insulin signaling and whole-body glucose regulation remain unclear. We discovered that high-fat feeding in rodents decreases the phosphorylation level of Dynamin-related Protein 1 (Drp1) in ser-637 thus causing its activation and the consequent increase of mitochondrial fission the DVC. This increase in mitochondria fission, in turn, increases iNOS levels, ER-stress (seen as an increase in p-PERK) and phosphorylation of IRS1 Ser-1101 that causes insulin resistance. Chemical inhibition of Drp1 by injecting Drp1 inhibitor MDIVI-1 in the DVC, inhibits mitochondrial fission, ER stress and phosphorylation of IRS1 Ser-1101 thus restoring DVC-insulin ability to lower hepatic glucose production in vivo. These results were also confirmed by expressing the dominant negative form of Drp1 in the DVC. Conversely, molecular activation of Drp1 in the DVC of healthy rodents is sufficient to induce DVC mitochondrial fission, ER stress as well as insulin resistance. Taken together, these data illustrate that Drp1-dependent mitochondrial fission in the DVC is sufficient and necessary to induce insulin resistance and to dysregulate hepatic glucose production, and suggest that targeting the Drp1-mitochondrial-dependent pathways in the brain may carry the therapeutic potential to reverse insulin resistance.