Nesfatin-1, derived from nucleobindin 2, was recently identified as an anorexigenic signal peptide. However, its neural role in glucose homeostasis and insulin sensitivity is unknown. To evaluate the metabolic impact and underlying mechanisms of central nesfatin-1 signaling, we infused nesfatin-1 in the third cerebral ventricle of high-fat diet (HFD)-fed rats. The effects of central nesfatin-1 on glucose metabolism and changes in transcription factors and signaling pathways were assessed during euglycemic-hyperinsulinemic clamping. The infusion of nesfatin-1 into the third cerebral ventricle markedly inhibited hepatic glucose production (HGP), promoted muscle glucose uptake, and was accompanied by decreases in hepatic mRNA and protein expression and enzymatic activity of PEPCK in both standard diet- and HFD-fed rats. In addition, central nesfatin-1 increased insulin receptor (InsR)/insulin receptor substrate-1 (IRS-1)/AMP-dependent protein kinase (AMPK)/Akt kinase (Akt)/target of rapamycin complex (TORC) 2 phosphorylation and resulted in an increase in Fos immunoreactivity in the hypothalamic nuclei that mediate glucose homeostasis. Taken together, these results reveal what we believe to be a novel site of action of nesfatin-1 on HGP and the PEPCK/InsR/IRS-1/AMPK/Akt/TORC2 pathway and suggest that hypothalamic nesfatin-1 action through a neural-mediated pathway can contribute to increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake in vivo.