Modulation of neuropeptide receptors is important for pancreatic β-cell function. Here, islet distribution and effects of the neurotensin (NT) receptor modulators, xenin and NT, was examined. Xenin, but not NT, significantly improved glucose disposal and insulin secretion, in mice. However, both peptides stimulated insulin secretion from rodent β-cells at 5.6 mm glucose, with xenin having similar insulinotropic actions at 16.7 mm glucose. In contrast, NT inhibited glucose-induced insulin secretion. Similar observations were made in human 1.1B4 β-cells and isolated mouse islets. Interestingly, similar xenin levels were recorded in pancreatic and small intestinal tissue. Arginine and glucose stimulated xenin release from islets. Streptozotocin treatment decreased and hydrocortisone treatment increased β-cell mass in mice. Xenin co-localisation with glucagon was increased by streptozotocin, but unaltered in hydrocortisone mice. This corresponded to elevated plasma xenin levels in streptozotocin mice. In addition, co-localisation of xenin with insulin was increased by hydrocortisone, and decreased by streptozotocin. Further in vitro investigations revealed that xenin and NT protected β-cells against streptozotocin-induced cytotoxicity. Xenin augmented rodent and human β-cell proliferation, whereas NT displayed proliferative actions only in human β-cells. These data highlight the involvement of NT signalling pathways for the possible modulation of β-cell function.