The lumen of the large intestine houses approximately 1013 -1014 microorganisms. In order to defend itself, the intestinal epithelium secretes a protective coating of mucus, the major component of which is Muc2 mucin protein. There is much interest in the mechanism of excitation-mucus secretion coupling because compromised mucus layer formation in Muc2 knock-out mice and human disease is associated with direct contact of bacteria with the epithelium, inflammation, bloody diarrhoea and colon cancer. Previously, cholinergic stimulation has been shown to promote mucus secretion, but the mechanism for excitation-mucus secretion coupling is not fully understood. The aim of the current study was to elucidate the mechanism of calcium (Ca2+) signal generation in native human colonic crypt goblet cells in situ and determine the role of Ca2+ signalling in Muc2 protein secretion. Methods: Human colonic crypts were isolated from colorectal tissue samples obtained at surgical resection (NRES approval) and placed in a 3D culture system. Intracellular Ca2+ was monitored by Fura2/Fluo-4 imaging. To complement previous qRT-PCR mRNA expression analyses, localisation of M3AChR, CD38 (which catalyses NAADP synthesis), TPC1&2 (i.e. Ca2+ channels stimulated by NAADP) and endolysosomes (LAMP-1 positive puncta) within Muc2+ goblet cells was visualised by fluorescence immunolabelling and confocal microscopy. Ca2+ signal generation and Muc2 depletion from goblet cells was assessed following stimulation with carbachol (Cch,10μM) in the presence and absence of nicotinamide (20 mM, an inhibitor of CD38-mediated NAADP synthesis) or NED-19 (200 μM, an NAADP receptor inhibitor). Results: Application of Cch evoked a Ca2+ signal at the human colonic crypt base which spread to all cell types located along the human colonic crypt-axis, including goblet cells. Muc2+ goblet cells expressed: M3AChRs on the basal membrane; CD38 in the cytoplasm and nucleus; cytoplasmic TPC1&2 associated with apical LAMP-1 positive intracellular puncta. Cch-induced colonic crypt Ca2+ signals were suppressed by pre-treatment with nicotinamide (64.5±5% reduction, N=2 subjects, n=5 crypts) and were inhibited profoundly by pre-incubation with NED-19 (85±5% reduction, N=5 subjects, n=15 crypts). Goblet cell Muc2 immunofluorescence was dramatically reduced following a 30 minute exposure to Cch (44±4.7 % reduction, N=4 subjects, n=4 crypts) and this was inhibited by NED-19 (150±45%, N=4 subjects, n=4 crypts). Conclusions: Goblets cells in the human large intestine express the molecular machinery to synthesise NAADP. Inhibition of the CD38-NAADP-TPC pathway suppressed Ca2+ signal generation and Muc2 depletion. The CD38-NAADP-TPC-calcium pathway plays a central role in cholinergic excitation-mucus secretion coupling in human colonic crypt goblet cells in situ.