The oral antidiabetic agent metformin acts at least partially via an activation of AMPK in liver and muscle cells. It has recently appeared that hypothalamic AMPK is a key regulator of feeding in mammals. Since metformin also exhibits anorectic effects in animal models as well as in humans, we hypothesized that AMPK may be a target of metformin in hypothalamic neurons. Here we show that in primary cultures of rat hypothalamic neurons, low glucose levels stimulate the phosphorylation of AMPK, thus increasing NPY gene expression. The addition of metformin in low glucose conditions was found to block AMPK phosphorylation. Consistently, the stimulation of NPY observed in low glucose conditions was also inhibited by the drug. POMC gene expression measured in parallel was inhibited under low glucose conditions, but in contrast to NPY, it was not dependent upon AMPK and not affected by metformin. Taken together, our data demonstrate that metformin can inhibit AMPK activity in hypothalamic neurons, thus modulating the expression of the orexigenic peptide NPY. These results provide for the first time a potential mechanism of action for the anorectic effects of metformin, a widely used drug that could represent a valuable adjunct to novel therapies aimed at modulating central feeding pathways.