Menthol-a natural organic compound-is widely used for relieving various pain conditions including migraine. However, a high dose of menthol reportedly decreases pain thresholds and enhances pain responses. Accordingly, in the present study, we addressed the effect of menthol on the excitability of acutely isolated dural afferent neurons, which were identified with a fluorescent dye, using the whole-cell patch-clamp technique. Under a voltage-clamped condition, menthol altered the holding current levels in a concentration-dependent manner. The menthol-induced current (I) remained unaffected by the addition of selective transient receptor potential melastatin 8 antagonists. Moreover, the reversal potential of I was similar to the equilibrium potential of K. I was accompanied by an increase in input resistance, thereby suggesting that menthol decreases the leak K conductance. Under a current-clamped condition, menthol caused depolarization of the membrane potential and decreased the threshold for the generation of action potential. While the I was substantially inhibited by 10 μM XE-991, a selective K7 blocker, the M-current mediated by K7 was not detected in the nociceptive neurons tested in the present study. Moreover, I decreased under acidic extracellular pH conditions or in the presence of 3 μM A-1899, a selective K2P3.1 and K2P9.1 blocker. The present results suggest that menthol inhibits leak K channels, possibly acid-sensitive two-pore domain K channels, thereby increasing the excitability of nociceptive sensory neurons. The resultant increase in neuron excitability may partially be responsible for the pronociceptive effect mediated by high menthol doses.