Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increased single channel activities of transient receptor potential ankyrin-1 (TRPA1) in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and which was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared with allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through desensitization of TRPA1-expressing nociceptors.