Understanding mechanisms by which glibenclamide stimulates insulin release is important particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Anti-diabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high affinity K_ATP channel receptors at the plasma membrane of cells. This normally occurs during glucose stimulation where ATP inhibition of plasmalemmal K_ATP channels leads to voltage-activation of L-type calcium channels for rapidly switching on and off calcium influx governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas including glibenclamide have additional K_ATP channel receptors within cells at insulin granules. We tested non-permeabilized cells in mouse islets for glibenclamide stimulated insulin secretion mediated by granule localized K_ATP channels by using conditions that bypass glibenclamide action on plasmalemmal K_ATP channels. High potassium stimulation evoked a sustained rise in cell calcium but a transient rise in insulin secretion. With continued high potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule localized K_ATP channels that functionally contribute to sustained second phase insulin secretion.