We recently showed that activation of GPR119 (also termed glucose-dependent insulinotropic receptor, or GDIR) improves glucose homeostasis via direct cAMP-mediated enhancement of glucose-dependent insulin release in pancreatic -cells. Here we show that GPR119 also stimulates incretin hormone release and thus may regulate glucose homeostasis by this additional mechanism. GPR119 mRNA was found to be expressed at significant levels in intestinal sub-regions that produce GIP and GLP-1. Furthermore, in situ hybridization studies indicated that most GLP-1-producing cells co-express GPR119 mRNA. In GLUTag cells, a well-established model of intestinal L-cell function, the potent GPR119 agonist AR231453 stimulated cAMP accumulation and GLP-1 release. When administered in mice, AR231453 increased active GLP-1 levels within two minutes after oral glucose delivery and substantially enhanced total GIP levels. Blockade of GLP-1 receptor signaling with exendin(9–39) reduced the ability of AR231453 to improve glucose tolerance in mice. Conversely, combined administration of AR231453 and the DPP-4 inhibitor sitagliptin to wild-type mice significantly amplified both plasma GLP-1 levels and oral glucose tolerance, relative to either agent alone. In mice lacking GPR119, no such enhancement was seen. Thus, GPR119 regulates glucose tolerance by acting on intestinal endocrine cells as well as pancreatic -cells. These data also suggest that combined stimulation of incretin hormone release and protection against incretin hormone degradation may be an effective anti-diabetic strategy.