Singlet oxygen (1O2) is the molecular oxygen in electronically excited state. 1O2 can be generated through photodynamic processes which require three elements: light, oxygen, and photosensitizer. Using the Xenopus oocyte expression system and the patch-clamp fluorometry setup, we discovered heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels on excised membrane patches are sensitive to the photodynamic modification (PDM) mediated by 1O2. 1O2 modification increased the voltage-insensitive current component and prolonged the deactivation of mouse HCN2 channel.  Then we extended this study to native HCN channels in thalamocortical (TC) neurons in the brain. FITC-cAMP was chosen as the photosensitizer and was delivered into the recorded neuron via whole-cell patch-clamp recording pipette. After illuminating the brain slice with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were loaded into the neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. Next, we investigated impacts of the PDM on the membrane excitability of TC neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential. Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. Therefore, PDM of native HCN channels can be developed as a unique and novel biophotonics approach to modulate neuronal excitability.