Allyl isothiocyanate (mustard oil, MO) is a highly reactive electrophilic compound known to cause irritation, pain and inflammation. These effects are thus far though to be mediated by activation of TRPA1, a Transient Receptor Potential (TRP) cation channel expressed in nociceptive neurons. Recent research has shown that TRPV1, the heat and capsaicin receptor, can be also activated by reactive compounds such as allicin and leek and onion extracts. Here, we show that both human and mouse TRPV1 are activated by MO, at concentrations at which TRPA1 undergoes fast desensitization and block. In Ca²⁺ imaging experiments of intact HEK293 cells, MO induces an increase of the intracellular Ca²⁺, which was not present when Ca²⁺ was omitted in the bath solution. Activation of TRPV1 by MO is dose-dependent and is caused by a shift of the voltage dependence of channel activation to more negative potentials, similar to the activation of TRPV1 by capsaicin. Stimulation of TRPV1 by MO can be observed in inside-out patches, indicating a membrane-delimited mechanism of activation. Notably, MO was able to stimulate a large population of sensory neurons isolated from Trpa1 KO mice. This population was significantly reduced in Trpa1/Trpv1 double KO mice, indicating the physiological importance of TRPV1 activation by MO. The response observed in the double KO mice is consistent with an activation of TRPM8. WT, Trpa1 and Trpv1 KO mice displayed significantly stronger aversion to MO than double KO mice in forced drinking and open field exploration assays. The identification of TRPV1 and TRPM8 as novel targets of MO is essential for the full understanding of the mechanisms of action of this compound in vivo and prompts to re-evaluate the results of previous research, in which MO was used as specific activator of TRPA1.