Traditionally seeds of Sisymbrium irio Linn has been used in different regions of Pakistan for the treatment of gastrointestinal, airways and vascular system ailments. To insight the pharmacological basis in vitro study was conducted in order to validate its folkloric uses in hypertension. 70% aqueous-methanolic extract of seeds from Sisymbrium irio (Si.MEs) was tested on isolated rabbit aorta, jejunum and trachea strip hanged in tissue bath having physiological solutions aerated with carbogen and their responses were measured and recorded via Power Lab. The Si.MEs exhibited the transient spasmogenic effect (0.01-1.0 mg/mL) on spontaneous jejunum contractions, followed by the spasmolytic effect. The addition of atropine resulted in blocking in spasmogenic effect while the spasmolytic effect was originated, suggesting the presence of an antimuscarinic effect. Likewise verapamil, Si.MEs (0.03-5 mg/mL) repressed the high concentration K + (80 mM)-induced contraction and also drifted the Ca 2+ concentration-response curves toward right (0.3-3.0 mg/mL), possibly signifying the Ca 2+ channel blockade. Furthermore, Si.MEs exhibited nonspecific relaxant effect on carbachol (1 μM)- and high concentration K + (80 mM)-induced tracheal contractions in a way comparable to dicyclomine, suggesting the coexistence of Ca 2+ antagonistic and/or antimuscarinic properties. Additionally, Si.MEs also relaxed the phenylephrine(1 μM)- and high concentration K + (80 mM)-induced aortic contraction (0.01-3 mg/mL), suggesting blockade of Ca 2+ channel. Moreover, oral administration of Si.MEs, as high as 6 g per kg, did not produce lethality among the treated groups of mice. Aqueous-methanolic extract of seeds from S. irio (Si.MEs) exhibited the bronchodilator and gut modulator (spasmogenic and spasmolytic) activities, probably through dual blockade of muscarinic receptors and Ca 2+ channels, whereas, vasodilator effect may be due to Ca 2+ channels blockade validates its use in hypertension.