Background: In traditional medicine, asafoetida obtained from the roots of different Ferula assafoetida (F. asafoetida) is used for various therapeutic purposes such as whooping cough, pneumonia and bronchitis in children, as well as asthma. Objectives: In this study, the effect of asafoetida on muscarinic receptors of tracheal smooth muscle, as one possible mechanism responsible for the relaxant effect seen for the plant, was evaluated. Materials and Methods:The effects of three cumulative concentrations of aqueous extract of F. asafoetida (2.5, 5 and 10 mg/mL), 10 nM atropine, and saline on muscarinic receptors were tested in tracheal smooth muscle samples by performing cumulative concentration response curve to methacholine (a muscarinic receptor agonist) and assessing the shift in concentration response curves due to different concentrations of the extract and atropine. Results: The EC 50 obtained in the presence of higher concentration of the extract muscle was significantly higher compared to saline (P < 0.01). The maximum responses to methacholine in the presence of higher concentration of the extract (10 mg/mL) was significantly lower than that of saline (P < 0.05). The values of CR-1, obtained in the presence of all concentrations of the extract, were significantly lower compared to atropine in the experimental group (P < 0.01 to P < 0.001). Conclusions: These results showed an inhibitory effect for the extract of asafetida on muscarinic receptors of tracheal smooth muscle.