Neuronostatin is a recently described peptide hormone encoded by the somatostatin gene. We previously showed that intra-peritoneal injection of neuronostatin into mice resulted in c-Jun accumulation in pancreatic islets in a pattern consistent with the activation of glucagon-producing alpha cells. We therefore hypothesized that neuronostatin could influence glucose homeostasis via a direct effect on the alpha cell. Neuronostatin enhanced low glucose-induced glucagon release in isolated rat islets and in the immortalized alpha cell line, αTC1-9. Furthermore, incubation with neuronostatin led to an increase in transcription of glucagon mRNA, as determined by RT-PCR. Neuronostatin also inhibited glucose-stimulated insulin secretion from isolated islets. However, neuronostatin did not alter insulin release from the beta cell line INS 832/13, indicating that the effect of neuronostatin on insulin secretion may be secondary to a direct action on the alpha cell. In agreement with our in vitro data, intra-arterial infusion of neuronostatin in male rats delayed glucose disposal and inhibited insulin release during a glucose challenge. These studies suggest that neuronostatin participates in maintaining glucose homeostasis through cell-cell interactions between α-cells and β-cells in the endocrine pancreas leading to attenuation in insulin secretion.