Peperomia hispidula (Sw.) A. Dietr. is used in Mexican traditional medicine for treating respiratory illnesses such as asthma. The latter disorder results from an excessive and inappropriate constriction of airway smooth muscle. The aim of the present study was to evaluate the relaxant activity of P. hispidula on isolated rat tracheal rings contracted with carbachol. The methyleugenol was identified as the main active constituent in the dichloromethane extract. To explore the possible mechanism of action, concentration-response curves were constructed in the presence and absence of propranolol (3 μM), indomethacin (10 μM), glibenclamide (1 μM), and L-NAME (300μM), finding that neither reduced methyleugenol-induced smooth muscle relaxation. In conclusion, P. hispidula herein displayed relaxant activity on rat tracheal rings. The effect of methyleugenol, was probably not related to the activation of β2-adrenoceptors, prostaglandins, K+ATP channels or nitric oxide.