The existence of functional connexin36 (Cx36) hemichannels in β-cells was investigated in pancreatic islets of rat and wild-type (Cx36^+/+), mono- (Cx36^+/-), and bi-allelic (Cx36^-/-) knockout mice. Hemichannel opening by KCl depolarization was studied measuring ATP release and changes of intracellular ATP (ADP). Cx36+/+ islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36^-/- than Cx36^+/+ islets and was not decreased by KCl depolarization; Cx36^+/- islets showed values between that of control and homozygous islets. 5 mM extracellular ATP increased ATP content, ATP/ADP ratio, and induced a biphasic insulin secretion in depolarized Cx36^+/+ and Cx36^+/- but not Cx36^-/- islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential and their activation was inhibited by mefloquine and glucose (IC50~8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet β-cells might avoid depolarization induced ATP loss allowing an optimum increase of the ATP /ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36^+/- and Cx36^-/- islets confirms that Cx36 gap-junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.