Peripheral serotonin (5-hydrodytryptamine; 5-HT) is involved in the regulation of gastrointestinal motility and sensation, whereas centrally it plays a role in mood regulation. A dysfunctional serotonergic system may provide a plausible link between functional dyspepsia symptoms and its high psychosocial comorbidity such as anxiety and depression. The aim of this study was to evaluate the effect of decreased 5-HT synthesis by acute tryptophan depletion (ATD) on gastric sensorimotor function and nutrient tolerance, anxiety scores, and gastrointestinal mucosal 5-HT concentrations in healthy volunteers. All subjects were studied under a control condition and during ATD. Gastric sensorimotor function and nutrient tolerance were assessed using a barostat (n = 16, mean age 28.8 ± 1.4 yr) and a satiety drinking test (n = 13, mean age 27.3 ± 1.4 yr). Anxiety during the barostat was evaluated using State-Trait Anxiety Inventory (STAI) questionnaire. 5-HT concentrations were measured in fundic and duodenal mucosal biopsies by means of ELISA and immunohistochemistry. ATD significantly decreased plasma tryptophan levels compared with control in every experiment. ATD did not affect gastric sensitivity and compliance but decreased the sensation of nausea during balloon distension (AUC: 17.4 ± 4.3 vs. 11.4 ± 3.4 mm·mmHg, P = 0.030). ATD enhanced the postprandial volume increase (ANOVA, P < 0.05), but this was not accompanied by augmented nutrient tolerance (848 ± 110 vs. 837 ± 99 ml, nonsignificant). ATD had no effect on STAI state anxiety scores. No evidence was found for an effect on the number of enterochromaffin cells, but ATD reduced 5-HT levels in the duodenal mucosa. ATD alters gastric postprandial motor function and distension-induced nausea. These findings confirm involvement of 5-HT in the control of gastric accommodation and sensitivity.