The human transient receptor potential melastatin 8 (hTRPM8) ion channel is a nonselective cation channel involved in human health and disease such as cancer, pain, obesity and diabetes. hTRPM8 has been shown to be gated in a polymodal manner by voltage, cold temperature, lipids, modulatory proteins, and chemical ligands. More specifically, hTRPM8 is activated by a number of cold inducing small molecules, including menthol and icilin. A better understanding of the molecular mechanism of ligand-gated channel activation may prove useful in unlocking the TRPM8 therapeutic potential. The sensing domain (transmembrane helices S1-S4) of TRPM8 has been implicated as the nexus of ligand binding, which is coupled to channel gating. Here, we present the direct binding of menthol to the isolated hTRPM8 sensing domain with nuclear magnetic resonance (NMR), circular dichroism (CD), and microscale thermophoresis (MST). We then compare the effects of binding to two previously identified residues, Y745 (on S2) and R842 (on S4), that have been shown to abrogate TRPM8 menthol sensitivity. The data presented indicate specific binding of menthol to the wild type as well as Y745H and R842H mutant hTRPM8 sensing domains, suggesting that menthol binding and the subsequent coupling to hTRPM8 channel gating are distinct processes.