This study was designed to evaluate the effect of jatrorrhizine on smooth muscle contractions isolated from rat ileum longitudinal muscles. Jatrorrhizine increased the amplitude of spontaneous contractions of ileum longitudinal muscles in concentration-dependent manner with an EC 50 of 30.0 ± 8.4 μM. Preincubation of ileum strips with atropine (1 μM), 4-diphenyllacetoxy- N (2-chloriethyl)-piperidine (4-DAMP, 1 μM) or darifenacin (1 μM) significantly inhibited acetylcholine (0.1 μM)- and jatrorrhizine (100 μM)-induced ileum longitudinal muscle contractions, whereas they were not affected by AF-DX116 (1 μM) or hexamethonium (100 μM). Pretreatment with SB204070 (1 μM) rather than 3-tropanyl-indole-3-carboxyleat (tropisetron, 1 μM) significantly inhibited 5-HT (10 μM)-induced ileum longitudinal muscle contractions. In contrast, jatrorrhizine-induced ileum longitudinal muscle contractions were not inhibited by tropisetron or SB204070. Furthermore, jatrorrhizine-induced ileum longitudinal muscle contractions were strongly inhibited by nifedipine (1 μM), and also attenuated by removal of extracellular Ca 2+, U73122 (1 μM), ruthenium red (50 μM) or 2-aminoethoxydiphenylborate (2-APB, 10 μM). Taken together, jatrorrhizine-elicited spontaneous contractions in rat ileum longitudinal muscles are mediated by activation of acetylcholine receptors, mostly the M 3 receptor. Ca 2+ influx through L-type Ca 2+ channel is significantly contributed to jatrorrhizine-elicited spontaneous contractions, and Ca 2+ release via IP 3 and ryanodine pathways are also involved.