Gut microbiota alterations have been described in several diseases with altered gastrointestinal (GI) motility, and awareness is increasing regarding the role of the gut microbiome in modulating GI function. Serotonin [5-hydroxytryptamine (5-HT)] is a key regulator of GI motility and secretion. To determine the relationship among gut microbes, colonic contractility, and host serotonergic gene expression, we evaluated mice that were germ-free (GF) or humanized (HM; ex-GF colonized with human gut microbiota). 5-HT reduced contractile duration in both GF and HM colons. Microbiota from HM and conventionally raised (CR) mice significantly increased colonic mRNAs Tph1 [(tryptophan hydroxylase) 1, rate limiting for mucosal 5-HT synthesis; P< 0.01] and chromogranin A (neuroendocrine secretion; P< 0.01), with no effect on monoamine oxidase A (serotonin catabolism), serotonin receptor 5-HT 4, or mouse serotonin transporter. HM and CR mice also had increased colonic Tph1 protein (P< 0.05) and 5-HT concentrations (GF, 17�3 ng/mg; HM, 25�2 ng/mg; and CR, 35�3 ng/mg; P< 0.05). Enterochromaffin (EC) cell numbers (cells producing 5-HT) were unchanged. Short-chain fatty acids (SCFAs) promoted TPH1 transcription in BON cells (human EC cell model). Thus, gut microbiota acting through SCFAs are important determinants of enteric 5-HT production and homeostasis.—Reigstad, CS, Salmonson, CE, Rainey, III, JF, Szurszewski, JH, Linden, DR, Sonnenburg, JL, Farrugia, G., Kashyap, PC Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells.