Peripheral serotonin (5-HT) from intestinal enterochromaffin cells (EEC) is an important regulator of gastrointestinal function, hepatic gluconeogenesis, adipose lipolysis and thermogenesis. Increases in plasma 5-HT link to poorer glycaemic control in subjects with type 2 diabetes (T2D), while polymorphisms in tryptophan hydroxylase (Tph1, which synthesises 5 HT) link with human obesity. We assessed glucose-stimulated 5-HT release from the colon and primary ECC in mice, from healthy lean and obese subjects and subjects with type 2 diabetic (T2D), and assessed duodenal Tph1 expression in these, and bariatric (RYGB) subjects. Glucose above 100 mM triggered 5-HT release from mouse colon, with augmented release from individual vesicles shown by single cell amperometry. Tph1 transcript expression was higher in T2D subjects (1.5-fold, P<0.05) and correlated with BMI in non-T2D subjects (P<0.05); transcripts were lower in RYGB compared to obese and T2D (P<0.05). Tph1 transcript levels were unaffected by glycaemic status in lean and T2D subjects, and tended to increase after luminal glucose only in T2D subjects (euglycemia 1.7-fold, P=0.09). Plasma 5-HT levels were higher in morbidly obese subjects with T2D at baseline (P<0.01) and after intraduodenal glucose (AUC, P<0.05); baseline levels correlated positively with BMI across all subjects (P<0.005). In conclusion (i) glucose-stimulated 5 HT release occurs via increased release from single vesicles, (ii) obese and T2D subjects may have increased gut-5 HT capacity, while (iii) gut 5-HT capacity may be lowered following RYGB. These findings support a key role of gut-5-HT in metabolic dysregulation in obesity and T2D.