Introduction: Mechanisms by which gut luminal content regulates secretion and motility is ill-understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal-sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Methods and Results: Pure (98-99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) GPCRs, and transporters for glutamine (SNAT1/2), glucose (GLUT1/3/SGLT1) and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5HT release (EC₅₀=0.002-0.2µM; 2-fold release) but were 10-100x more potent in neoplastic EC cells which also secreted 6-13x more 5HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol/eugenol), also stimulated normal and neoplastic EC cell 5HT secretion (EC₅₀=1.2nM-2.1µMand 0.05nM-0.1µM release, respectively) while 2-deoxyglucose and the artificial sweetener, sucralose, stimulated (EC₅₀=9.2nM and 0.38nM). 5HT release was associated with ERK phosphorylation (1.5-fold, p<0.02) and could be inhibited by a somatostatin analogue (IC₅₀: 10^-12M). Eleven secretory associated genes including the vesicle docking inhibitor, STXBP3, were up-regulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression using antisense knockdown significantly (p<0.05) reduced 5HT secretion. Conclusions: EC cells express GPRCs and transporters for luminal tastants, olfactants, glutamine, glucose and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation and 5HT secretion. Luminal EC cell regulation is likely to be as important as the G cell regulation in gastric acid secretion; development of agents to target EC cell function is a critical therapeutic goal.