•  Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the body's total serotonin (5‐HT). •  Although 5‐HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level. •  This study provides the first functional characterisation of single primary guinea‐pig and human EC cells. •  EC cells release 5‐HT from large dense core vesicles in a calcium‐dependent manner with kinetics surprisingly resembling release from synaptic vesicles. •  3D modelling indicates that the quantity of 5‐HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5‐HT receptors. •  These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control. Abstract  The major source of serotonin (5‐HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5‐HT, and that this 5‐HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5‐HT release from single primary EC cells. We have developed a rapid primary culture of guinea‐pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca2+ imaging, immunocytochemistry and 3D modelling. Ca2+ enters EC cells upon stimulation and triggers quantal 5‐HT release via L‐type Ca2+ channels. Real time amperometric techniques reveal that EC cells release 5‐HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5‐HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5‐HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three‐dimensional (3D) simulations of 5‐HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5‐HT release in the whole‐tissue milieu. These models indicate that local 5‐HT levels are likely to be maintained around the activation threshold for mucosal 5‐HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5‐HT release in primary EC cells. The mode of 5‐HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.