TRPA1 is a non-selective cation channel and has been shown to be activated by a wide variety of noxious compounds and physiological stressors. Interestingly, TRPA1 is reported to be stimulated by menthol, an agonist of TRPM8, with different dose dependencies between mouse and human. It has been suggested that this different reactivity may be attributable to three amino acid residues in the TM5 region of TRPA1, namely, S–T–V in human and S–T–G in mouse. In this study, to further investigate the importance of the TM5 region through comparison with other mammalian species, canine TRPA1 cDNA was cloned and its reactivity to several agonists was compared in dog and mouse by using recombinant proteins. The TM5 region of cloned canine TRPA1 showed high similarity to that of human TRPA1 and the S–T–V residues were conserved. HEK293T cells were transfected with mouse or canine TRPA1 and subjected to calcium influx imaging. Both mouse and canine TRPA1 were activated by the TRPA1 agonist allyl isothiocyanate and were inactivated by the TRPA1 antagonist HC-030031. In contrast, reactivity to menthol was observed to differ between these two species. Mouse TRPA1 was activated by 100 µM of menthol and showed transient Ca²⁺ influx when menthol was washed out (“off response”), whereas canine TRPA1 activation required a high (300 µM) concentration of menthol, but no off response was observed. These results showed that the reactivity of canine TRPA1 against menthol is similar to that of human TRPA1 but not to mouse TRPA1, which might reflect the similarity of their respective TM5 regions.