Mentha longifolia is grown commercially for production of oils and leaves. For centuries, this plant has widely been used traditionally as a folk remedy for treatment of human and ruminant gastrointestinal complaints in the Northwest of Iran. The essential oil of the plant was extracted using hydrodistillation. In the present study a total of 12 constituents, representing 96.6% of the oil were identified by gas chromatography/mass spectrometry (GC/MS). The main compounds were 1,8-cineole (19.8%), pulegone (17.3%), caryophyllene oxide (14.8%) and isomenthone (12.4%). The effects of 0.1 to 1000 g/mL essence of Mentha longofolia (EOML) on ruminal and abomasal smooth muscle of 24 healthy sheep were assessed in vitro. In the isolated ruminal and abomasal preparations, EOML (0.1â100 g/mL) exhibited a weak spasmogenic effect followed by relaxation and complete (p < 0.05) abolition of the spontaneous contraction at the highest dose (1000 g/mL). In contrast rat ileum only showed a dose-dependent relaxation effect, and tissues preincubated with EOML decreased the acetylcholine (ACh)-induced contraction. EOML (1000 g/mL) significantly inhibited the effect of ACh suggesting that the effect may be mediated via cholinergic receptors on smooth muscle. These primary data indicated that the plant contained spasmogenic and spasmolitic constituents. Moreover the results showed that the EOML profoundly alters gastrointestinal smooth muscle contraction in a dose-dependent and tissue-specific manner. The results suggested that EOML has the potential for development as a prokinetic and relaxant agent that may prevent or alleviate dysfunctions of gastrointestinal motility.