Transient receptor potential ankyrin 1 (TRPA1) is a Ca²⁺-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca²⁺ response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca²⁺ response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca²⁺ response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca²⁺ response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca²⁺ response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca²⁺ response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX. © 2014 the American Physiological Society.