Glucagon-like peptide-1 (GLP-1) released from intestinal endocrine L cells after food intake, is a potent glucose-dependent stimulant of insulin secretion. Plasma levels of GLP-1 rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve. Our previous studies showed the involvement of M1 muscarinic receptors expressed by the L cells in the regulation of postprandial GLP-1 secretion in rats. The goal of this study was to explore the involvement of muscarinic receptors in the regulation of GLP-1 secretion by human L cells using a newly described human L cell line (NCI-H716). PMA (positive control) stimulated GLP-1 secretion to 252 ± 38% of the control (P < 0.001). Bethanechol, a non-selective muscarinic agonist, significantly stimulated GLP-1 secretion to 187 ± 20% of the control (P < 0.01, n = 8). Pirenzepine (M1 antagonist; 10 to 1000 µM) and gallamine (M2 antagonist; 10 to 1000 µM) completely inhibited bethanechol-induced GLP-1 secretion, while 4-diphenylacetoxy-N-methylpiperidine (M3 antagonist) had no effect on bethanechol-stimulated GLP-1 secretion. McN-A-343 (M1 muscarinic agonist) dose-dependently stimulated GLP-1 secretion (to 252 ± 50% of control at 1000 µM; P < 0.01), while oxotremorine (M3 agonist) had no effect. M1, M2 and M3 muscarinic receptors were shown to be expressed in NCI-H716 cells by Western blot, immunohystochemistry and RT-PCR. Expression of the M1, M2 and M3 muscarinic receptor subtypes was also confirmed in paraffin-embedded human small intestine sections by double immunofluorescent staining. These results demonstrate the role of M1 and M2 muscarinic receptors expressed by human L cells in the control of GLP-1 secretion.