Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst₅ KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst₅ were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst₅ KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst₅/sst₁-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst₅ KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst₅ KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst₅ KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst₅ mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst₅ antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.