Aim: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. Methods: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. Results: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. Conclusion: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance.