The homotetrameric thermosensitive transient receptor potential vanilloid 1-4 (TRPV1-4) channels in sensory neurons are strongly responsive to heat stimuli. However, their cold activations have not been reported in line with the nonzero heat capacity difference during heat or cold unfolding transitions. Here, along with the experimental examinations of the predicted ring size changes in different domains against the central pore during channel gating at various temperatures, the K169A mutant of reduced human TRPV3 was first found to be activated and inactivated by cold below 42°C. Further thermoring analyses revealed distinct heat and cold unfolding pathways, which resulted in different protein thermostabilities. Thus, both cold and heat unfolding transitions of thermosensitive TRPV1-4 channels may exist once a mutation destabilizes the closed state.