We hypothesized that citrate might modulate the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK)/(ACC) pathway and participate in neuronal feeding control and glucose homeostasis. To address this issue, we injected citrate into the lateral ventricle of rats. Intracerebroventricular (ICV) injection of citrate diminished the phosphorylation of hypothalamic AMPK/ACC, increased the expression of anorexigenic neuropeptide (pro-opiomelanocortin and corticotropin-releasing hormone), elevated the level of malonyl-CoA in the hypothalamus, and reduced food intake. No change was observed in the concentration of blood insulin after the injection of citrate. With a euglycemic-hyperinsulinemic clamp, the glucose infusion rate was higher in the citrate group than in the control group (28.6+/-0.8 vs 19.3+/-0.2 mU/kg body weight/min respectively), and so was glucose uptake in skeletal muscle and the epididymal fat pad. Concordantly, insulin receptor (IR), IR substrate type 1 (IRS1), IRS2, and protein kinase B (AKT) phosphorylation in adipose tissue and skeletal muscle was improved by citrate ICV treatment. Moreover, the treatment with citrate for 7 days promoted body weight loss and decreased the adipose tissue. Our results suggest that citrate and glucose may serve as signals of energy and nutrient availability to hypothalamic cells.