Transient receptor potential vanilloid 3 (TRPV3) channel robustly expressed in the skin is nonselective and multimodal cation channel that is activated by warm temperature, voltage and chemicals. We recently identified a natural monoterpenoid carvacrol that specifically activates TRPV3 channels. However, how the carvacrol activates TRPV3 remains unknown. Here, we report the specific activation of TRPV3 by carvacrol in assays of both whole-cell and single-channel recordings. To understand the molecular mechanism underlying TRPV3 activation by carvacrol, we started model the interaction between carvacrol and cryo-EM TRPV3 structure using molecular docking approach. Site-directed mutagenesis further confirms that three key residues in the binding pocket formed primarily by the S2-S3 loop that is specific for allosteric activation of TRPV3 by carvacrol, but not agonist 2-APB. Our findings demonstrate a direct action on TRPV3 target by natural carvacrol that can be used as a specific tool for study of TRPV3 pharmacology.