BACKGROUND: Trachyspermum ammi is a plant of umbelliferae family and has been traditionally used in the treatment of many gastrointestinal disorders such as indigestion, colic, and diarrhea. Our previous study demonstrated that aqueous extract of Trachyspermum ammi reduced the contractile activity of rat's ileum. AIMS: This study was designed to examine the effect of Trachyspermum ammi essence on acetylcholine-induced contraction in isolated rat's ileum. MATERIALS AND METHODS: In this experimental procedure, the tissue samples were suspended from the transducer lever and mounted on the organ bath containing Tyrode's solution. Isotonic responses were recorded by using an isotonic transducer and the amplitude of contractions. This process was induced by cumulative logarithmic concentrations of acetylcholine before and after exposing tissue by saline and different concentrations of essence. RESULTS: The chemical constituents of the essence from distillate extract of Trachyspermum ammi seeds were studied by gas chromatography mass spectrometry (GC-MS) and the chemical analysis showed many components in which thymol was the main constituent. Our findings showed that the essence derived from the extract in all concentrations used in this study (0.002, 0.005, and 0.01% V/V) significantly reduced acetylcholine-induced contractions (47.34, 60.46, and 86%, respectively, P < 0.05). The same concentrations of the essence also exhibit a significant anti-spasmodic action on acetylcholine-induced contractions (0.1,88.3, and 90.7%, respectively, P < 0.05). CONCLUSION: Since thymol was the major constituent of our samples of Trachyspermum ammi, the relaxant effect observed in this study is probably due to this agent which can exert an anti-cholinergic property.