Crotamiton (N–ethyl–o–crotonotoluidide) has long been used as an anti–itch agent. However, the mechanism by which crotamiton exerts anti–itch effects is unknown. Based on recent studies showing that transient receptor potential (TRP) channels are involved in itch sensations, we hypothesized that crotamiton could affect the activity of TRP channels. In this study, we found that crotamiton strongly inhibits TRPV (vanilloid) 4 channel activity. Crotamiton also inhibited itch–related behaviors induced by the TRPV4–selective agonist GSK1016790A. In patch–clamp experiments we observed large TRPV4 currents following crotamiton washout. In this washout current, single–channel open probabilities and unitary current amplitudes of TRPV4 were increased, which together were suggestive of TRPV4 pore dilation. To explore whether TRPV4 pore dilation occurred, we performed cation replacement experiments in which whole–cell currents and reversal potentials were measured. Our observation of increased cation influx and changes in reversal potentials upon crotamiton washout indicated the presence of TRPV4 pore dilation. These results identified TRPV4 as a molecular target of crotamiton and demonstrated pore dilation of TRPV4 upon crotamiton washout.