Sweet taste receptor subunits and α-gustducin found in enteroendocrine cells of the small intestine have been implicated in release of the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) in response to glucose and noncaloric sweeteners. α-Gustducin has also been found in colon, although its function there is unclear. We examined expression of α-gustducin, GLP-1, and GIP throughout the intestine. The number of α-gustducin-expressing cells and those coexpressing α-gustducin together with GLP-1 and/or GIP increased from small intestine to colon. α-Gustducin also was coexpressed with fatty acid G protein-coupled receptor (GPR) 40, GPR41, GPR43, GPR119, GPR120, and bile acid G protein-coupled receptor TGR5 in enteroendocrine cells of the colon. In colon, GPR43 was coexpressed with GPR119 and GPR120, but not with TGR5. Treatment of colonic mucosa isolated from wild-type mice with acetate, butyrate, oleic acid, oleoylethanolamide, or lithocholic acid stimulated GLP-1 secretion. However, GLP-1 release in response to these fatty acids was impaired in colonic tissue from α-gustducin knockout mice.