Peripheral and spinal hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels play important roles in neuropathic pain by regulating neuronal excitability. However, the participation of HCN channels in the ventral-lateral periaqueductal gray (vlPAG) during neuropathic pain states has not been clarified. To investigate the role of vlPAG HCN channels in neuropathic pain, the authors developed a chronic constriction injury (CCI) model. By using western blot analysis, they detected the upregulation of HCN1 and HCN2 channel expression at vlPAG 14 days post-CCI surgery. Subsequently, the function of these upregulated channels was verified by the intravlPAG infusion of ZD7288, a specific HCN blocker, which significantly relieved mechanical allodynia and thermal hyperalgesia in CCI animals. These results suggest that the upregulation of vlPAG HCN channels plays an important role in pain maintenance and might be a target for attenuating pain.