Objective To elucidate the molecular mechanism of the negative regulation of inflammatory factors by menthol through transient receptor potential melastatin 8 (TRPM8). Methods In an in vivo experiment, we detected expression of the cold-sensitive receptor TRPM8, nuclear transcription factor NF-κB, and inflammatory factor TNF-α after intravenous injection of (-)-menthol. In an in vitro experiment, we examined the effect of menthol on TRPM8, NF-κB, and TNF-α expression in PC12 cells. Finally, we examined the negative relationship between TRPM8 and TNF-α in PC12 cells (TRPM8 knockdown). Results The three main findings of this study are as follows. First, after intravenous injection of menthol, expression of the cold-sensitive receptor TRPM8 in the hypothalamus of mice was activated, and expression of the nuclear transcription factor NF-κB and inflammatory factor TNF-α was inhibited. Second, in the neuron-like PC12 cells, menthol activated TRPM8 expression and inhibited NF-κB and TNF-α. Third, in PC12 cells with TRPM8 knockdown, menthol showed no significant effect on TRPM8 expression; in contrast, TNF-α expression was activated. Conclusions The negative regulation of inflammatory factors by menthol depends on the activation of TRPM8 expression.