Nutrient sensors mediating the stimulatory effects of food-derived carbohydrates and lipids on incretin and insulin secretion have been widely studied. However, mechanisms underlying amino acid regulation of those processes remain poorly understood. GPR142 was recently reported to be highly expressed in pancreatic beta cells and selectively activated by L-Tryptophan (L-Trp). Moreover, synthetic GPR142 agonists were demonstrated to stimulate glucose-dependent insulin secretion and improve glucose tolerance in vivo. In addition to pancreatic islets, GPR142 is also expressed in the gastrointestinal tract. However, whether GPR142 is involved in regulation of incretin release from this tissue and required for sensing of naturally occurring amino acids remains unclear. In this study, we have confirmed that L-Trp potentiates glucose-dependent insulin secretion from pancreatic islets and improves glucose tolerance in vivo, and these effects were blunted in GPR142 knockout mice (KO). Moreover, we found that oral dosing of L-Trp or the literature GPR142 agonist compound A (CpdA) increased plasma levels of incretin hormones, such as GIP and GLP-1, and these effects were absent in KO mice. Noteworthy, meal-induced elevation in plasma incretins and insulin was also reduced in KO mice. Together, our data indicate that GPR142 acts as a nutrient sensor and is critically required for L-Trp to stimulate release of insulin and incretins. Thus, GPR142 agonists may represent a novel class of therapeutic agents that leverages amino acid sensing pathways for the treatment of type 2 diabetes.