Background and PurposeThe mechanism of the anti‐migraine action of extracts of butterbur [Petasites hybridus(L.) Gaertn.] is unknown. Here, we investigated the ability of isopetasin, a major constituent of these extracts, to specifically target TRPA1 channel and to affect functional responses relevant to migraine.Experimental ApproachSingle‐cell calcium imaging and patch‐clamp recordings in human and rodent TRPA1‐expressing cells, neurogenic motor responses in rodent isolated urinary bladder, release of CGRP from mouse spinal cordin vitroand facial rubbing in mice and meningeal blood flow in rats were examined.Key ResultsIsopetasin induced (i) calcium responses and currents in rat/mouse trigeminal ganglion (TG) neurons and in cells expressing the human TRPA1, (ii) substance P‐mediated contractions of rat isolated urinary bladders and (iii) CGRP release from mouse dorsal spinal cord, responses that were selectively abolished by genetic deletion or pharmacological antagonism of TRPA1 channels. Pre‐exposure to isopetasin produced marked desensitization of allyl isothiocyanate (AITC, TRPA1 channel agonist)‐ or capsaicin (TRPV1 channel agonist)‐evoked currents in rat TG neurons, contractions of rat or mouse bladder and CGRP release from mouse central terminals of primary sensory neurons. Repeated intragastric administration of isopetasin attenuated mouse facial rubbing, evoked by local AITC or capsaicin, and dilation of rat meningeal arteries by acrolein or ethanol (TRPA1 and TRPV1 channel agonists respectively).Conclusion and ImplicationsActivation of TRPA1 channels by isopetasin results in excitation of neuropeptide‐containing nociceptors, followed by marked heterologous neuronal desensitization. Such atten uation in pain and neurogenic inflammation may account for the anti‐migraine action of butterbur.