TRPM8 antagonists derived from its cognate ligand, (−)-menthol, are underrepresented. We determine the absolute stereochemistry of a well-known TRPM8 antagonist, (−)-menthyl 1, using VCD and 2D NMR. We explore 1 for its antagonist effects of the human TRPM8 (hTRPM8) orthologue to uncover species-dependent inhibition versus rat channels. (−)-Menthyl 1 inhibits menthol-and icilin-evoked Ca 2+ responses at hTRPM8 with IC 50 values of 805 ± 200 nM and 1.8 ± 0.6 μM, respectively, while more potently inhibiting agonist responses at the rat orthologue (rTRPM8 IC 50 (menthol) = 117 ± 18 nM, IC 50 (icilin) = 521 ± 20 nM). Whole-cell patch-clamp recordings of hTRPM8 confirm the 1 inhibition of menthol-stimulated currents, with an IC 50 of 700 ± 200 nM. We demonstrate that 1 possesses ≥400-fold selectivity for hTRPM8 versus hTRPA1/hTRPV1. (−)-menthyl 1 can be used as a novel chemical tool to study hTRPM8 pharmacology and differences in species commonly used in drug discovery.