Of the four syntaxins specialized for exocytosis, syntaxin (Syn)-2 is the least understood. In this study, we used Syn-2/epimorphin knockout mice to examine the role of Syn-2 in insulin secretory granule (SG) exocytosis. Unexpectedly, Syn-2 knockout mice exhibited paradoxical superior glucose homeostasis resulting from an enhanced insulin secretion. This was confirmed in vitro by pancreatic islet perifusion showing an amplified biphasic glucose-stimulated insulin secretion arising from an increase in size of the readily releasable pool of insulin SGs and enhanced SG pool refilling. The increase in insulin exocytosis was attributed mainly to an enhanced recruitment of the larger pool of newcomer SGs that undergoes no residence time on plasma membrane before fusion and, to a lesser extent, also the predocked SGs. Consistently, Syn-2 depletion resulted in a stimulation-induced increase in abundance of exocytotic complexes we previously demonstrated as mediating the fusion of newcomer SGs (Syn-3/VAMP8/SNAP25/Munc18b) and predocked SGs (Syn-1A/VAMP2/SNAP25/Muncn18a). This work is the first to show in mammals that Syn-2 could function as an inhibitory SNARE protein that, when relieved, could promote exocytosis in pancreatic islet β-cells. Thus, Syn-2 may serve as a potential target to treat diabetes.