Betagranin, a N-terminal fragment of chromogranin A (CgA), results from a proteolytic processing, and is co-secreted with insulin. While other CgA-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic ß-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are downregulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line, and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.