In view of the importance of molecular sensing in the function of the gastrointestinal (GI) tract, we assessed whether signal transduction proteins that mediate taste signaling are expressed in cells of the human gut. Here, we demonstrated that the α-subunit of the taste-specific G protein gustducin (Gα_gust) is expressed prominently in cells of the human colon that also contain chromogranin A, an established marker of endocrine cells. Double-labeling immunofluorescence and staining of serial sections demonstrated that Gα_gust localized to enteroendocrine L cells that express peptide YY and glucagon-like peptide-1 in the human colonic mucosa. We also found expression of transcripts encoding human type 2 receptor (hT2R) family members, hT1R3, and Gα_gust in the human colon and in the human intestinal endocrine cell lines (HuTu-80 and NCI-H716 cells). Stimulation of HuTu-80 or NCI-H716 cells with the bitter-tasting compound phenylthiocarbamide, which binds hT2R38, induced a rapid increase in the intracellular Ca²⁺ concentration in these cells. The identification of Gα_gust and chemosensory receptors that perceive chemical components of ingested substances, including drugs and toxins, in open enteroendocrine L cells has important implications for understanding molecular sensing in the human GI tract and for developing novel therapeutic compounds that modify the function of these receptors in the gut.