Pheretima (family Megascolecidae) has been documented as a potent agent for the treatment of cough and breathing difficulty in traditional Chinese medicine for nearly 2000 years. The water extract of Pheretima was separated into three fractions of the ethanolic precipitate, the alkaline fraction and the acidic fraction. Among the three fractions, the acidic fraction showed the most potent spasmolytic effects on histamine-induced contractions in isolated guinea pig tracheal rings, and the most inhibitory activities on increase of short circuit current induced by carbachol in isolated rat tracheal epitheliums with the IC50 values of 0.15 and 0.08 mg/ml, respectively. Further in vivo studies also displayed that the acidic fraction could protect experimental asthma model induced by the combination of histamine and acetylcholine chloride in guinea pigs to prolong the latent periods of asthma (P < 0.05) and significantly decrease the cough frequency caused by ammonia water in mice (P < 0.001).