The mechanisms by which glucose regulates the activity of glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH) are largely unknown. We have previously shown that AMP-activated protein kinase (AMPK) increases nitric oxide (NO) production in VMH GI neurons. We hypothesized that AMPK-mediated NO signaling is required for depolarization of VMH GI neurons in response to decreased glucose. In support of our hypothesis, inhibition of neuronal nitric oxide synthase (nNOS) or the NO receptor soluble guanylyl cyclase (sGC) blocked depolarization of GI neurons to decreased glucose from 2.5 to 0.7mM or AMPK activation. Conversely, activation of sGC or the cell permeable analog of cGMP, 8-Br-cGMP, enhanced the response of GI neurons to decreased glucose suggesting that stimulation of NO-sGC-cGMP signaling by AMPK is required for glucose sensing in GI neurons. Interestingly, the AMPK inhibitor Compound C completely blocked the effect of sGC activation or 8-Br-cGMP and 8-br-cGMP increased VMH AMPK2 phosphorylation. These data suggest that NO, in turn, amplifies AMPK activation in GI neurons. Finally, inhibition of the cystic fibrosis transmembrane regulator (CFTR) Cl- conductance blocked depolarization of GI neurons to decreased glucose or AMPK activation, while decreased glucose, AMPK activation and 8-Br-cGMP increased VMH CFTR phosphorylation. We conclude that decreased glucose triggers the following sequence of events leading to depolarization in VMH GI neurons: AMPK activation, nNOS phosphorylation, NO production, stimulation of sGC-cGMP signaling which amplifies AMPK activation and leads to closure of the CFTR.