Amino acid availability is monitored by animals to adapt to their nutritional environment. Beyond gustatory receptors and systemic amino acid sensors, enteroendocrine cells (EECs) are believed to directly percept dietary amino acids and secrete regulatory peptides. However, the cellular machinery underlying amino acid-sensing by EECs and how EEC-derived hormones modulate feeding behavior remain elusive. Here, by developing tools to specifically manipulate EECs, we find that Drosophila neuropeptide F (NPF) from mated female EECs inhibits feeding, similar to human PYY. Mechanistically, dietary L-Glutamate acts through the metabotropic glutamate receptor mGluR to decelerate calcium oscillations in EECs, thereby causing reduced NPF secretion via dense-core vesicles. Furthermore, two dopaminergic enteric neurons expressing NPFR perceive EEC-derived NPF and relay an anorexigenic signal to the brain. Thus, our findings provide mechanistic insights into how EECs assess food quality and identify a conserved mode of action that explains how gut NPF/PYY modulates food intake.|Enteroendocrine cells in the gut sense food to regulate feeding behavior. Here, the authors show in Drosophila L-glutamate reduces the frequency of calcium oscillations in enteroendocrine cells and decreases the release of NPF, thereby reducing the activation of a pair of anorexic enteric neurons.