© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group.Objectives: The calcium-sensing receptor (CaSR), the major sensor of extracellular Ca2+, is expressed in various tissues, including the gastrointestinal tract. Although the essential ligand of CaSR is calcium, its activity can be regulated by aromatic L-amino acids. The expression of CaSR on enteroendocrine cells suggests that CaSR functions as a physiological amino acid sensor for gut hormone release. Here, we investigated the effects of L-tryptophan (L-Trp) on rat glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and insulin secretion, and the role of CaSR in this mechanism in vivo. Methods: The effects of intraduodenal L-Trp on GLP-1, PYY, and insulin secretion were investigated. A CaSR antagonist, NPS 2143, was administered to determine whether CaSR plays a role in L-Trp-mediated gut hormone release. Male Wistar rats were divided into L-Trp, L-Trp+NPS 2143, and L-Trp+vehicle groups. Blood samples were collected, before and after the intraduodenal infusions, for determining plasma glucose, L-Trp, insulin, GLP-1, and PYY levels. Results: Our study showed a significant increase in plasma GLP-1 and insulin levels, but not plasma PYY and glucose levels, following the acute intraduodenal administration of L-Trp. We demonstrated that CaSR plays a role in L-Trp-mediated GLP-1 secretion due to attenuation of GLP-1 release with the CaSR antagonist NPS 2143. Discussion: We demonstrated that GLP-1, but not PYY, secretion following intraduodenal L-Trp administration was mediated through calcium-sensing receptors. This mechanism underlying protein sensing in the gastrointestinal system may be important for the development of new therapeutic strategies without side effects for obesity and diabetes.