Introduction The hypothalamus is implicated in nervous regulation of the glucose homeostasis. Detection of increased blood glucose level by specific hypothalamic glucose sensitive neurons triggers physiologic responses such as increase insulin secretion and decreased food intake. Astrocytes are suspected to be involved in brain glucose-sensing. They present a network organization formed by numerous gap-junctions (GJ) of connexins. This allows transfer of glucose from bloodstream to neurons. The main connexin expressed in astrocytes is the connexin 43 (Cx43). We hypothesized that the Cx43-dependant astrocyte networks plays a critical role in hypothalamic glucose-sensing. Materials and Methods Expression of Cx43 was studied by immunochemistry in rat ventro-median hypothalamus (VMH). Decrease in VMH Cx43 expression was assessed, in vivo, by stereotaxic injection of siRNA against Cx43. Evaluation of hypothalamic glucose-sensing was assessed by monitoring insulin secretion or refeeding of 20h-fasted animals in response to an intracarotid injection of a glucose bolus towards the brain. Results Cx43 is highly expressed in the VMH at the astrocytic end-feet, around blood vessels. Inhibition of Cx43 (about 30%) leads to a decrease of food intake 72h after the injection, whitout modification of weigth, blood glucose and insulin levels. Intracarotid glucose injection-induced insulin secretion is significantly decreased in siCx43 rats. Similarly, the satietogenic effect of glucose is significantly attenuated in 20h-fasted siCx43 rats during the refeeding. Conclusion These results show that Cx43-dependant astrocyte networks are critical for hypothalamic glucose sensitivity.