Atractylenolide I (8,9-dehydroasterolide, B), 4,15-epoxy-8 beta-hydroxyasterolide (C) and atractylenolide III (8 beta-hydroasterolide, D) from Atractylodes macrocephala Koidz, 28.0, 56.0 mumol/L, inhibited the spontaneous movement of rat isolated ileum with contractile force decreased obviously (P < 0.001), but the rest force not affected (P > 0.05). B, C and D inhibited the spasm enhanced by Ach and His to rat ileum, rightly shifted the dose-response curves and reduced the maximal response (P < 0.05). Pretreatment with B, C or D 28.0, 56.0 mumol/L Ver 0.28 mumol/L significantly antagonized CaCl2-induced contraction of rat isolated ileum. Co bathing of B, C or D 56.0 mumol/L with Atr 0.19 mumol/L weakened the response of ileum to Ach. B, C or D 56.0 mumol/L restrained the spasm of ileum mediated by Neo 2.15 mumol/L. The modified Phenol Red Paste Model demonstrated that B, C or D in dose of 28.0 mg/kg inhibited the gastrointestinal prochoresis of mice significantly. These results suggested that B, C and D inhibit the gastrointestinal movement of rat and the spasm induced by the agonists. Their activities are related to the inhibition of cholinergic system and Ca2+.