Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (−/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (−/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (−/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the β-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 β-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the β-cells and not the α-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic β-cells.