Crataegus sp. has been used in the traditional medicine of Mexico as well as other countries for the treatment of several respiratory diseases, such as flu, cough and asthma. The tracheal relaxant effect of the leaves of C. mexicana is investigated here for the first time, through a bioassay-guided study by using isolated tracheal rings of guinea-pig as an experimental model. The hexane extract was the most active compared to dichloromethane and methanol. An active fraction was obtained from the hexane extract. Assays by HPLC-MS reveal that at least 14 compounds may exist in it. In addition, the results suggest that relaxant effect of the effective fraction was in part related to the activity of β-adrenergic receptors and not to K+ AT P channels. This study represents the first in which the relaxant effect of leaves of C. mexicana on tracheal rings of guinea pig was clearly demonstrated. More studies are required to correctly identify the bioactive compounds that contribute to the relaxant effects of Crataegus mexicana, and to know the mechanisms of action of these compounds.