Oxytocin (OT) is an anorexic agent. Deficits in OT are associated with human obesity. Glucokinase (GK), a hallmark of glucose-sensing cells, is expressed in the supraoptic nucleus (SON) of the hypothalamus, where OT and vasopressin (VP) are synthesized. Therefore, SON neurons may function as glucose-sensors. It has been shown previously that an increase in glucose induced a rise in intracellular calcium ([Ca++]i) in SON neurons (Song and Sladek, 2011), presumably through GK-driven production of ATP and subsequent closure of ATP dependent potassium channels (KATP). In our present studies, live-cell calcium imaging was thus used to determine the molecular signaling mechanisms underlying glucose-sensing of SON neurons. Rat hypothalamic explants that include SON were loaded with the calcium sensitive dye, Fura-2AM. Changes in [Ca++]i were monitored by changes in the 340/380 emission in response to an increase in the glucose concentration from 0.5 to 5 mM in the presence of KCl (13 mM, to increase resting membrane potential). This increment in glucose caused an increase in [Ca++]i in the majority of SON neurons. The responses still existed in the presence of TTX (3 uM, to block the influence of afferent synaptic inputs) but were abolished when cells were pretreated with either alloxan (4 mM, to inhibit GK activity) or diazoxide (0.4 mM, to open KATP channels). These findings indicate that SON neurons are capable of sensing glucose increase and respond to it by ATP-driven closure of KATP channels, which in turn induces an increase in [Ca++]i and thus will potentially alter OT hormone release. This is the first demonstration that SON neurons monitor extracellular glucose, thus supports a role for OT in maintaining body nutrient homeostasis.