Temperature-sensitive two-pore domain potassium channels (thermal K2P) are recently shown to cluster at nodes of Ranvier (NRs) and play a key role in action potential (AP) regeneration and conduction on Ab-afferent nerves. Cooling temperatures affect AP regeneration and conduction on Ab-afferent nerves but the underlying mechanisms are not completely understood. Here, we have performed patch-clamp recordings directly at the NR in an ex vivo tri-geminal nerve preparation. We have characterized the effects of cooling temperatures on intrinsic electrophysiological properties and AP regeneration at the NR on rat Ab-afferent nerves, and determined whether and how thermal K2P channels may be involved in the effects of cooling temperatures. We show that cooling temperatures from 35°C to 15°C decrease outward leak currents, increase input resistance, depolarize resting membrane potential (RMP), broad-en AP width and increase latency of AP threshold at the NR. We further demonstrate that cooling temperatures im-pair regeneration of high-frequency AP trains at the NR. The effects of cooling temperatures on the intrinsic electrophysiological properties and regeneration of high-frequency AP trains at the NR can be partially reversed by BL-1249 (BL), arachidonic acid (AA), and intra-axonal protons, three thermal K2P activators, indicating the involvement of thermal K2P channels. Moreover, we show that at cooling temperatures there are interplays among thermal K2P channels, RMPs, and voltage-gated Na1 channels, which together limit regeneration of high-frequency AP trains at the NR. Our findings demonstrate a new role of thermal K2P channels in temperature-dependent conduction of high-frequency sensory signals.