Serotonin (5-hydroxytryptamine, 5-HT), an important neurotransmitter and a paracrine messenger in the gastrointestinal (GI) tract, regulates intestinal secretion by its action primarily on 5-HT₃ and 5-HT₄ receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study we determine if human-derived gut microbiota affect host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age matched Swiss Webster germ free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short circuit current (Isc) in GF compared to HM mice. Additionally, 5-HT₃ receptor mRNA and protein expression was significantly higher in GF compared to HM mice. Ondansetron, a 5-HT₃ receptor antagonist, inhibited 5-HT evoked Isc in GF mice but not in HM mice. Furthermore, a 5-HT₃ receptor selective agonist, 2-methyl 5-hydroxytryptamine hydrochloride evoked a significantly higher Isc in GF compared to HM mice. Immunohistochemistry in 5-HT3A-GFP mice localized 5-HT₃ receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT evoked Isc between GF and HM mice persisted in the presence of tetrodotoxin (TTX), but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT₃ receptor mRNA in GF mice colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT₃ receptor expression via acetate production. Epithelial 5-HT₃ receptor may function as a mediator of gut microbiota driven change in intestinal secretion.