Peptide YY (PYY) is released following food intake and regulates intestinal function and glucose homeostasis, but the mechanisms underpinning these processes are unclear. Enteroendocrine L cells contain PYY and express the acylethanolamine receptor, Gpr119. Here, we show that Gpr119 activation inhibited epithelial electrolyte secretion in human and mouse colon in a glucose-sensitive manner. Endogenous PYY selectively mediated these effects, since PYY^−/− mice showed no Gpr119 response, but responses were observed in NPY^−/− mice. Importantly, Gpr119 responses in wild-type (WT) mouse tissue and human colon were abolished by Y₁ receptor antagonism, but were not enhanced by dipeptidylpeptidase IV blockade, indicating that PYY processing to PYY(3-36) was not important. In addition, Gpr119 agonism reduced glycemic excursions after oral glucose delivery to WT mice but not PYY^−/− mice. Taken together, these data demonstrate a previously unrecognized role of PYY in mediating intestinal Gpr119 activity and an associated function in controlling glucose tolerance.