Background: Betahistine is used as an H3 antagonist. It has been used to treat balance disorders. During the administration of the drug, the trachea may be affected through oral intake. Aim: This study aimed to determine the effects of betahistine on the tracheal smooth muscle of rats in vitro. Methods: On a rat trachea that had been isolated and immersed in Krebs solution in a muscle bath, we evaluated the efficacy of betahistine. We examined how the application of parasympathetic mimetic agents altered tracheal contractility. The betahistine was evaluated using the following criteria: the drug’s effects on tracheal smooth muscle contractions triggered by parasympathetic mimetic 10 − 6 M methacholine, electrically induced tracheal smooth muscle contractions, and resting tracheal smooth muscle tension were listed below. Results: At preparation concentrations as high as 10 − 4 M, betahistine produced a substantial relaxing response. The medication also prevented spike contraction brought by electrical field stimulation. However, betahistine alone had a negligible effect on the basal tension of the trachea at increasing concentrations. Conclusion: According to this study, excessive levels of betahistine might actually oppose cholinergic receptors and prevented the tracheal smooth muscles parasympathetic activity.