Traditional natural plants have been used throughout the world for their antidiabetic effects. The aim of the present study was to investigate the stimulating activity of a polysaccharide extract derived from T. aestivum sprout (TASP) on insulin secretion in vitro using the RIN-5F pancreatic β-cell line and rat pancreatic islets. In these experiments, TASP (0.1 to 2 mg/ml) augmented glucose-stimulated insulin secretion in a dose-dependent manner in the presence of a stimulatory glucose concentration (16.7 mM), but not of a basal concentration (1.1 mM). Although TASP failed to enhance the high K+-induced insulin secretion, the insulinotropic effect of TASP was significantly inhibited by diazoxide, an opener of ATP-sensitive K+ channel blocking insulin release. TASP potentiated the insulin secretion induced by other secretagogues, such as IBMX and tolbutamide. Moreover, glucose-derived blood insulin levels were significantly elevated by oral administration of TASP to mice, similarly to antidiabetic drugs. We also demonstrated that TASP significantly increased glucose-induced 45Ca2+ uptake and proinsulin mRNA expression in rat islets. Overall, our results suggest that TASP has a stimulating effect on insulin secretion and production in pancreatic β-cells via K+ channel closure and calcium influx. These results suggest that TASP may be useful as a candidate for the therapy of diabetes mellitus.