This study explored the mechanisms underlying the effectiveness of Vitex negundo in hyperactive respiratory disorders. Crude extract of V. negundo leaves was obtained. For in vivo bronchodilatory activity in anesthetized rats, different doses (1, 3, 10, 30, and 50 mg/kg) of the crude extract of V. negundo (Vn.Cr) were tested. The underlying mechanisms were studied in isolated guinea pig tracheal strips, suspended in organ baths at 37 °C. Intravenous doses of the crude extract of Vn.Cr showed dose-dependent bronchodilatory effect (9-50%) against carbachol (CCh; 100 µg/kg)-induced bronchoconstriction, similar to aminophylline. In isolated guinea-pig tracheal strips, Vn.Crrelaxed CCh (1 µM) and high K(+) pre-contractions with respective EC50 values of 0.72 (0.48-1.10; n = 5) and 3.38 mg/mL (1.84-6.21; n = 4), similar to papaverine. Diltiazem also relaxed both contractions with more potency against high K(+) pre-contraction (p < 0.05). Pre-incubation of the tracheal strips with Vn.Cr potentiated the isoprenaline inhibitory concentration response curves (CRCs), similar to papaverine. The inhibitory effect against CCh and high K(+) suggests involvement of phosphodiesterase (PDE) inhibitory pathway(s), in addition to an inhibitory effect on Ca(++) entry. This finding was further strengthened when pre-treatment of the tracheal strips potentiated the isoprenaline CRCs. RESULTS suggest Vn.Cr possesses a combination of papaverine-like PDE inhibitor and diltiazem-like Ca(++) entry blocking constituents, which partly explain its bronchodilatory effect, thus validating its medicinal importance in asthma.