Mustard oil (MO, allyl isothiocyanate) is a highly electrophylic compound known to produce irritation, inflammation and pain. As such, it is used frequently in research and its effects were initially thought to be solely mediated by activation of the cation channel TRPA1. In contrast to this view, we have recently shown that MO also acts as an agonist of the capsaicin and heat receptor TRPV1, but the underlying molecular mechanisms remain unknown. We have found that MO effects on TRPV1 were rapidly reversible upon washout and were not significantly altered by the mutation of the residue C155, a residue which is essential for the action of the electrophile allicin. Hence, MO action on TRPV1 seems not to be mediated by long lasting covalent modification of cysteine residues. Notably, MO seems to act in a similar way as capsaicin, causing a shift of the voltage dependence of channel activation to more negative potentials. We therefore tested MO effects on TRPV1 mutants known to alter the sensitivity to capsaicin. We found that the substitution S512Y in the putative transmembrane domain 3 significantly reduces the ability of MO to induce stimulation and to shift of the voltage dependence of channel activation. In addition, we found that, similar to capsaicin, MO cross sensitize with other TRPV1 stimulators such as heat and extracellular protons. The molecular determinants of these interactions may help to understand the mechanisms of action of MO in real conditions (physiological temperatures and acidosis), as well as the mechanisms of MO-induced hyperalgesia to heat application.