Spexin is a peptide whose action is poorly understood but which is expressed in many tissues. This encouraged us to investigate the potential role of spexin in the regulation of pancreatic secretion. Cells/islets were incubated with different concentrations of glucose and spexin to measure insulin secretion. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and BrdU (5-bromo-2'-deoxyuridine) tests were performed to assess the viability and proliferation of pancreatic islets after spexin treatment. Real-time polymerase chain reaction was used to detect messenger RNA expression for insulin, insulin receptor, and Pdx (pancreatic duodenal homeobox-1). Insulin secretion from cultured cells and isolated islets was reduced by spexin at 16 mM glucose level. In obese rats, insulin secretion was decreased after injection with spexin. Spexin treatment showed an increase in cultured cells and pancreatic islets cell viability and proliferation as well as an increase in proliferating cell nuclear antigen protein level. In contrast, a decrease in insulin and Pdx gene expression was found. The effects of spexin on insulin secretion in vitro and in vivo and also on cells viability and proliferation confirm that this peptide may be strongly involved in the pathogenesis of diabetes or its recovery.