Abstract: Objective To investigate the analgesic mechanism of xylazine by inhibiting the activation of hyperpolarized cyclic nucleotide-gated (HCN) ion channels. Methods HCN subchannel 1 (HCN1) knockout mice (HCN1-/-) and HCN1 wild type mice (HCN1+/+) were intraperitoneally injected with physiological saline and xylazine (10, 20, 30, and 40 mg/kg). Mechanical pain test and tail flick test were used to test the analgesic effect of xylazine by using the percentage of the maximal possible effect (%MPE); The control group and test groups of different concentrations of xylazine (12.5, 25, 50, and 100 μmol/L) were set up using HEK 293 cells transfected HCN1 plasmid and HCN subchannel 2 (HCN2) plasmid, respectively. The activated current of hyperpolarized HEK 293 cells expressing HCN1 and HCN2 ion channels and the inhibition rate of xylazine on hyperpolarization-activated currents were recorded using a whole cell patch clamp. Results The results demonstrated that %MPE of the mechanical pain stimuli test and the thermal radiation stimuli test increased with the higher concentration of xylazine injected for both HCN1+/+ mice and HCN1-/- mice. When injecting xylazine by 30 mg/kg and 40 mg/kg, the %MPE of mechanical pain stimuli test for HCN1-/- mice were %MPE=（62.06±14.72）% and %MPE=（69.92±16.09）%, respectively; and the percentages of tail flick tests were （52.50±1.97）% and %MPE=（64.74±6.34）%, respectively. But for HCN1+/+ mice, the percentages of mechanical pain stimuli test were %MPE=（75.47±8.06）% and %MPE=（86.35±11.31）%; respectively, and the percentage of tail flick tests were %MPE=（57.83±4.82）% and （74.98±9.35）%. The analgesic effect results of the mechanical pain test and tail flick test of HCN1+/+ mice were significantly different from HCN1-/- mice ( P V1/2 of HCN1 was between （-79.58±1.56） mV-（-98.95±3.57） mV. The Vh inhibition rate of HCN2 by xylazine (12.5-100 μmol/L) was between （29.19±17.82）%-（80.02±6.64）%; with V1/2 of HCN2 between （-102.17±1.36） mV-（-117.48±2.38） mV. Conclusion Xylazine showed better analgesic effect on HCN1+/+ mice than HCN1-/- mice. Xylazine can produce analgesic effect by inhibiting HCN ion channel currents.