Background and PurposeItching is the most frequent pathology in dermatology that has significant impacts on people's mental health and social life. Transient receptor potential vanilloid 3 (TRPV3) channel is a promising target for treating pruritus. However, few selecetive and potent antagonists have been reported. This study was designed to identify selective TRPV3 antagonist and elucidate its anti‐pruritus pharmacology.Experimental ApproachFlexStation and calcium fluorescence imaging were conducted to track the functional compounds. Whole‐cell patch clamp was used to record itch‐related ion channel currents. Homologous recombination and site‐directed mutagenesis were employed to construct TRPV3 channel chimeras and point mutations for exploring pharmacological mechanism. Mouse models were used for in vivo anti‐pruritus assay.Key ResultsAn acridone alkaloid (citrusinine‐II) was purified and characterized from Atalantia monophylla. It directly interacts with Y564 within S4 helix of TRPV3 to selectively inhibit the channel with a half maximal inhibitory concentration (IC50) of 12.43 μM. Citrusinine‐II showed potential efficacy to attenuate both chronic and acute itch. Intradermal administration of citrusinine‐II (143 ng/skin site) nearly completely inhibited itch behaviours. It also shows significant analgesic effects. Little side effects of the compound are observed.Conclusion and ImplicationsBy acting as a selective and potent inhibitor of TRPV3 channel, citrusinine‐II shows valuable therapeutic effects in pruritus animal models and is a promising candidate drug and/or lead molecule for the development of anti‐pruritus drugs.