Feeding behavior is a complex process that depends on the ability of the brain to integrate hormonal and nutritional signals, such as glucose. One glucosensing mechanism relies on the glucose transporter 2 (GLUT2) in the hypothalamus, especially in radial glia-like cells called tanycytes. Here, we analyzed whether a GLUT2-dependent glucosensing mechanism is required for the normal regulation of feeding behavior in GFAP-positive tanycytes. Genetic inactivation of Glut2 in GFAP-expressing tanycytes was performed using Cre/Lox technology. The efficiency of GFAP-tanycyte targeting was analyzed in the anteroposterior and dorsoventral axes by evaluating GFP fluorescence. Feeding behavior, hormonal levels, neuronal activity using c-Fos, and neuropeptide expression were also analyzed in the fasting-to-refeeding transition. In basal conditions, Glut2-inactivated mice had normal food intake and meal patterns. Implementation of a preceeding fasting period led to decreased total food intake and a delay in meal initiation during refeeding. Additionally, Glut2 inactivation increased the number of c-Fos-positive cells in the ventromedial nucleus in response to fasting and a deregulation of Pomc expression in the fasting-to-refeeding transition. Thus, a GLUT2-dependent glucose-sensing mechanism in GFAP-tanycytes is required to control food consumption and promote meal initiation after a fasting period.