Menthol is an organic compound derived from mint oils, known for its cooling and soothing properties, often used in various pharmaceutical, cosmetic, and therapeutic applications. It exerts beneficial effects on the digestive system by relaxing smooth muscles, reducing spasms, and improving gastrointestinal motility. This study aimed to evaluate the effects of menthol on bovine ileal smooth muscle contractions in vitro. Ileal tissue segments were collected from adult cattle at an abattoir and subsequently immersed in 25 mL organ baths containing Tyrode’s solution. The baths were maintained at 37 °C and continuously aerated with a gas mixture of 95% O₂ and 5% CO₂. The tissues were subjected to various contractile agents, including potassium chloride (KCl) at concentrations of 30 and 80 mM, carbachol (CCh) at 1 μM and 4 μM, and barium chloride (BaCl₂) at 30 mM. Menthol was cumulatively applied in incremental concentrations to assess its modulatory effects on contraction amplitude. Results demonstrated that menthol elicited a dose-dependent inhibition of smooth muscle contractions across most stimulatory conditions, with the extent of inhibition varying among different stimuli. The Ca2+ channel blocking activity was further confirmed when pre-treatment of isolated ileums with menthol (23 and 200 μg mL⁻¹) caused a rightward shift in the Ca2+ concentration-response curves (CRCs), similar to verapamil. These findings suggest menthol’s spasmolytic action may be mediated through the modulation and inhibition of calcium channels. Menthol effectively attenuates bovine ileal smooth muscle contractions in vitro, indicating its potential as a natural therapeutic agent for controlling gastrointestinal hyperactivity in cattle.