Intestinal epithelial cells form a vital selective barrier between the mucosal immune system and a barrage of microorganisms, ligands and antigens derived from the hostile gut lumen. Preservation of barrier function is underpinned in part by calcium-dependent secretion of a protective mucus layer. The aims of the current study were to (i) unravel the mechanism of cholinergic calcium signals that initiate at the human colonic crypt base and (ii) determine the effects on secretion of mucus and fluid from crypt-base-goblet-cells GCs) and neighbouring intestinal stem cells (ISCs). Methods: Human colonic crypts were isolated from colorectal tissue samples obtained at surgical resection (NREC approval) and cultured as crypts in the short term (< 1 week), or propagated as crypt-like organoids over the long-term (up to 5 years). The spatio-temporal characteristics of intracellular calcium was monitored by Fura-2/Fluo-4/Calbryte-630 imaging and the mechanism of receptor-mediated calcium mobilisation was characterised by pharmacological and knockdown gene approaches. Calcium signalling toolkit expression was visualised by fluorescence immunolabelling and super-resolution imaging. Mucus secretion was visualised by Muc2 immunofluorescence depletion assays and real-time imaging of fluorescently tagged mucin-2, using MUC2::mNEON crypt-like organoids generated by CRISPR-HOT. An organoid swelling assay was used as a proxy for fluid secretion.  Results: A microdomain of juxtaposed InsP3R3 and TPC1 expression was present at the apico-lateral pole of ISCs and GCs at the crypt (or organoid crypt-like)-base and corresponded to the site of cholinergic calcium signal initiation (Carbachol, 1-100 µM; n>10). Calcium signals propagated across to the basal pole of initiation cells and laterally to neighbouring cells. Calcium signal amplitude was reduced >50% by TPC1 antagonists NED19 (250 µM; n>10; P<0.05) and tetrandrine (20 µM; n>10; P<0.05). Caffeine (10 mM), an inhibitor of InsP3Rs, and miRNA knockdown of InsP3R3s, also reduced calcium signal amplitude by >50% (n>5; P< 0.05).  Carbachol (10 µM) stimulated both MUC2 depletion from GCs in colonic crypt bases (n>200; P<0.05) and luminal secretion of MUC2-mNEON in crypt-like organoids (n>5; P<0.05).  Carbachol (10 µM) also stimulated an increase in organoid cross-sectional area in organoid swelling assays.  Pharmacologic inhibition of TPC1 or InsP3Rs (see above) reduced  cholinergic stimulation of: MUC2 immunofluorescence depletion from crypt GCs; luminal secretion of MUC2-mNEON from crypt-like organoids; and cross-sectional area of organoids in swelling assays (n>5; P<0.05). Conclusion: Co-activation of juxtaposed TPC1 and InsP3R3 is required for generation of cholinergic calcium signals and downstream secretion of hydrated mucus, which culminates in the flushing of the human colonic stem cell niche.