Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain and diarrhea. Since luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor FFA2 in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion (DBS) and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg, sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released into the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND+PA1) dose-dependently induced duodenal erosions. IND+PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG0974, ondansetron or omeprazole, but not by RS23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with ~8X more release at 0.1 mM than at 1 µM, the effects inhibited by co-perfusion of GLPG0974. Luminal PA1 at 1 µM increased, but at 0.1 mM diminished duodenal blood flow. Co-superfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment, but restored by ondansetron. These results suggest that although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic COX inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia.