The effect of Mentha longifolia (L.) leaf hydroalcoholic extract (MLE) was examined on rat ileal smooth muscle contractions. Last portion of ileum from male adult Wistar rat was mounted in an organ bath containing Tyrode solution. The tissue was contracted by carbachol (CCh, 10 μM), KCl (60 mM) and BaCl2 (4 mM) and then MLE (0.0625-1 mg mL-1) was added to the bath cumulatively. The effect of MLE on KCl-induced contraction was examined after tissue incubation with propranolol (1 μM), naloxone (1 μM) and Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM). The effect of MLE on CaCl2-induced ileal contraction in Ca2+-free with high potassium Tyrode solution was also evaluated. The role of potassium channels was examined by ileum incubation (5 min) with tetraethylammonium (TEA, 1 mM). The results showed that KCl-, CCh and BaCl2-induced ileal contractions were inhibited (p<0.001) by cumulative concentrations of MLE with the same potency. In addition, MLE (0.25-1 mg mL-1) inhibited (p<0.01) ileal contractions induced by CaCl2 (0.45-2.7 mM) in a concentration-related manner. The antispasmodic effect of MLE was affected neither by propranolol, L-NAME nor by naloxone. The MLE concentration-response curve was shifted to the right (p<0.05) by tissue incubation with TEA. From results it may be suggested that Mentha longifolia hydroalcoholic leaf extract induces its spasmolytic activity mainly through disturbance in calcium mobilization and partly by potassium channels activation. Present results show that Mentha longifolia leaf extract exerts relaxant effects on intestinal smooth muscle, consistent with the traditional use of the plant to treat gastrointestinal disorders such as diarrhea and colic.