The lipid sensor GPR119 is highly expressed by enteroendocrine L-cells and pancreatic β-cells that release the hormones, PYY and GLP-1, and insulin, respectively. Endogenous oleoylethanolamide (OEA) and the dietary metabolite, 2-monoacylglycerol (2-OG) can each activate GPR119. Here we compared mucosal responses to selective, synthetic GPR119 agonists (AR440006, AR231453) and the lipids, OEA, 2-OG and N-oleoyldopamine (OLDA) monitoring epithelial ion transport as a read-out for L-cell activity in native mouse and human gastrointestinal (GI) mucosae. We also assessed GPR119 modulation of colonic motility in wild-type (WT), GPR119-/- and PYY-/- mice.The water-soluble GPR119 agonist, AR440006 (that cannot traverse epithelial tight-junctions) elicited responses when added apically or basolaterally in mouse and human colonic mucosas. In both species, GPR119 responses were PYY, Y1 receptor-mediated and glucose-dependent. AR440006 efficacy matched the GI distribution of L-cells in WT tissues but was absent from GPR119-/- tissue. OEA and 2-OG responses were significantly reduced in GPR119-/- colon, but OLDA responses were unchanged. Alternative L-cell activation via free fatty acid receptors FFA1, FFA3, FFA4, the bile acid receptor TGR5, or by melanocortin MC4 were unchanged in GPR119-/- tissues. GPR119 agonism slowed transit in WT, but not PYY-/- colon in vitro. AR440006 (i.p.) slowed WT colonic and upper GI transit significantly in vivo.This data indicates that luminal or blood borne GPR119 agonism can stimulate L-cell PYY release with paracrine consequences and slower motility. We suggest that this glucose-dependent L-cell response to a gut-restricted GPR119 stimulus has potential therapeutic advantage in modulating insulinotropic signalling with reduced risk of hypoglycemia.