Neprilysin, a widely expressed peptidase upregulated in type 2 diabetes, is capable of cleaving and inactivating the insulinotropic glucagon-like peptide-1 (GLP-1). Like dipeptidyl peptidase-4 (DPP-4), inhibition of neprilysin activity under diabetic conditions is associated with increased active GLP-1 levels and improved glycemic control. While neprilysin expression has been demonstrated in islets, its local contribution to GLP-1-mediated insulin secretion remains unknown. We investigated in vitro whether islet neprilysin inhibition enhances insulin secretion in response to glucose and/or exogenous GLP-1, and whether these effects are mediated by GLP-1 receptor (GLP-1R). Further, we compared the effect of neprilysin versus DPP-4 inhibition on insulin secretion. Isolated islets from wild-type (Glp1r+/+) and GLP-1 receptor knockout (Glp1r-/-) mice were incubated with or without the neprilysin inhibitor thiorphan and/or the DPP-4 inhibitor sitagliptin for 2.5 hours. During the last hour, insulin secretion was assessed in response to 2.8 mmol/l or 20 mmol/l glucose alone or plus exogenous active GLP-1. In Glp1r+/+ islets, neprilysin inhibition enhanced 2.8 mmol/l and 20 mmol/l glucose- and GLP-1-mediated insulin secretion to the same extent as DPP-4 inhibition. These effects were blunted in Glp1r-/- islets. In conclusion, inhibition of islet neprilysin in vitro increases glucose-mediated insulin secretion in a GLP-1R-dependent manner and enhances the insulinotropic effect of exogenous active GLP-1. Thus, neprilysin inhibitors may have therapeutic potential in type 2 diabetes by preserving islet-derived and circulating active GLP-1 levels.