Cell membranes consist of heterogeneous lipid domains that influence key cellular processes, including signal transduction, endocytosis, and electrical excitability. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we found that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. The size of OMDs is also dependent on cholesterol levels and the structure of lipid tails. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This increased neuronal firing could be partially due to an increased open probability of HCN channels. In animal models of neuropathic pain, we observed shrunken OMDs and relocalization of HCN channels from OMDs to disordered lipid domains. The gating effect on HCN channels was likely a result of direct modulation of the voltage sensor by OMDs. These findings suggest that disturbances in lipid domains play a role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.