Our previous studies have shown that the anti-asthma Traditional Chinese Medicine herbal formula ASHMI inhibits acetylcholine-induced contractions of tracheal rings from ovalbumin-sensitized and naïve mice in a β-adrenoceptor independent manner. We sought to determine whether acute in vivo ASHMI administration inhibits airway hyperreactivity (AHR) in a murine model of allergic asthma and acetylcholine induced tracheal ring constriction ex vivo and to elucidate the cellular mechanisms underlying these effects. Ovalbumin-sensitized mice received a single oral ASHMI dose 2 hours prior to intravenous acetylcholine challenge. AHR was determined by invasive airway measurements. Myography was used to determine the effects of ASHMI on acetylcholine-induced constriction of tracheal rings from asthmatic mice with or without epithelial denudation. The effect of cyclooxygenase inhibition and EP2/EP4 receptor blockade on ASHMI attenuation of acetylcholine contractions were evaluated. Tracheal cAMP and PGE₂ levels were measured by ELISA. A single acute oral dose of ASHMI dramatically reduced AHR in response to acetylcholine provocation in ovalbumin-sensitized mice (P<0.001). In ex vivo experiments ASHMI significantly and dose-dependently reduced tracheal ring constriction to acetylcholine (P<0.05-0.001) which was epithelium-independent and associated with elevated cAMP levels. This effect was abrogated by cyclooxygenase inhibition or EP2/EP4 receptor blockade. ASHMI also inhibited contraction to high K+ (P<0.001). ASHMI increased tracheal ring PGE2 release in response to acetylcholine or high K+ (P<0.05 for both). ASHMI produced direct and acute inhibition of AHR in vivo, blocked acetylcholine-induced tracheal ring constriction via the EP2/EP4 receptor pathway identifying the mechanism by which ASHMI is an orally active bronchoprotective agent.