Over the last five decades, it has been suggested that glucose availability may regulate food intake. Despite the considerable efforts invested in unraveling the molecular mechanisms related to hypothalamic glucosensing and satiety response, there are still many unanswered questions. Here, we show that injection of an adenovirus expressing short hairpin RNA (shRNA) targeting GK into the 3V generates specific transduction of ependymal glia, especially tanycytes. GK knockdown animals showed no variation in NPY and AgRP (orexigenic neuropeptides) and CART (anorexigenic). POMC (another anorexigenic neuropeptide) showed to be even decreased in response to glucose when GK was knocked down. In conclusion, knockdown animals lost the normal response to glucose (i.e. a decrease in orexigenic neuropeptides and an increase in anorexigenic neuropeptides). In addition, alterations in feeding behavior (i.e., decreased satiety) were detected. Based on these results, we postulate that hypothalamic GK activity could regulate food intake as part of a glucostatic control system.