Menthol has a cooling effect via TRPM8 activation resulting in a relaxation, an anti-inflammation and an analgesic by the inhalation and the topical application. Previously, we demonstrated that menthol induced cytotoxicity in different ways dependent on its concentrations in lung cancer cell line, A549. However, the detail mechanisms of the cytotoxic action are unclear. The expression of molecules related to cytotoxicity was detected by using western blotting. Flow cytometrical analyses were performed to detect apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential. The expressions of some cyclins and cyclin-dependent kinases were suppressed by menthol (1 mM). At 2 mM, menthol evoked apoptosis Ca²⁺-independently concomitant with an increase of intracellular levels of ROS. N-acetyl cysteine failed to inhibit the menthol-induced apoptosis. Menthol rapidly decreased the mitochondrial membrane potential. These results suggest that menthol induces ROS-independent cytotoxicity despite the ROS production. Dysfunction of mitochondria may be involved in apoptotic action of menthol in A549.