The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal‐glial cells, termed tanycytes. The latter express glucose‐sensing proteins, including glucose transporter 2, glucokinase, and ATP‐sensitive K⁺ (K_ATP) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose‐induced rise of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cultured β‐tanycytes was examined. Fura‐2AM time‐lapse fluorescence images revealed that glucose increases the intracellular Ca²⁺ signal in a concentration‐dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K_ATP channel‐dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y₁ receptors followed by inositol trisphosphate receptor activation and Ca²⁺ release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca²⁺]_i in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes. � 2011 Wiley Periodicals, Inc.