TRPA1 channels have been found to play an important role in mammalian pain sensation, especially when the pain is caused by chemicals on site of inflammation. A large number of structurally diverse chemicals are found to activate TRPA1 channels, implicating a potential chemosensor in neuronal nociception. Identification of the channel activation by cysteine modification through covalent chemical reaction provides arguments for the diversity of the agonist structures. However, it is largely unknown how nonreactive compounds activate TRPA1 channels. Here, we report that NPPB, a classic Cl(-) channel blocker, potently activated human TRPA1 channels overexpressed in mammalian HEK-293 cells. This effect was confirmed in Ca(2+) imaging assay, patch clamp whole cell and single channel recordings. The NPPB response was quick, fully reversible and replicable, contrary to the effect of covalent modification by AITC. The mutagenesis studies revealed a refreshed look at several mutations known to be critical for the actions of AITC and menthol. The blocking profile of NPPB on these mutants showed that the NPPB activation was similar to that of FTS and different from AITC and menthol. The results indicated a possible close interaction between S5 and N-terminal domains of the channel. We also tested a group of NPPB analogs on TRPA1 channel activities. The results demonstrated that NPPB activation was tightly associated with chemical structure. None of the single chemical group was sufficient to activate the channel, indicating that NPPB activated TRPA1 through a structure-specific mechanism.