Background: Enteroendocrine cells collectively constitute our largest endocrine tissue, with serotonin (5-HT) secreting enterochromaffin (EC) cells being the largest component (∼50 %). This gut-derived 5-HT has multiple paracrine and endocrine roles. EC cells are thought to act as nutrient sensors and luminal glucose is the major absorbed form of carbohydrate in the gut and activates secretion in an array of cell types. It is unknown whether EC cells release 5-HT in response to glucose in primary EC cells. Furthermore, fasting augments 5-HT synthesis and release into the circulation. However, which nutrients cause fasting-induced synthesis of EC cell 5-HT is unknown. Here we examine the effects of acute and chronic changes in glucose availability on 5-HT release from intact tissue and single EC cells. Methods: We utilised established approaches in our laboratories measuring 5-HT release in intact mouse colon with amperometry. We then examined single EC cells function using our published protocol in guinea-pig colon. Single cell Ca2+ imaging and amperometry were used with these cells. Real-time PCR was used along with amperometry, on primary EC cells cultured for 24 h in 5 or 25 mM glucose. Results: We demonstrate that acute increases in glucose, at levels found in the gut lumen rather than in plasma, trigger 5-HT release from intact colon, and cause Ca2+ entry and 5-HT release in primary EC cells. Single cell amperometry demonstrates that high glucose increases the amount of 5-HT released from individual vesicles as they undergo exocytosis. Finally, 24 h incubation of EC cells in low glucose causes an increase in the transcription of the 5-HT synthesising enzyme Tph1 as well as increasing in 5-HT secretion in EC cells. Conclusions: We demonstrate that primary EC cells respond to acute changes in glucose availability through increases in intracellular Ca2+ the activation of 5-HT secretion, but respond to chronic changes in glucose levels through the transcriptional regulation of Tph1 to alter 5-HT synthesis.