Neuropathic pain is a devastating neurological disease that seriously affects patients' quality of life. Despite a high level of incidence, the underlying mechanisms of neuropathic pain are still poorly understood. However, recent evidence supports the prominent role of spinal glial cells in neuropathic pain states. In our laboratory, we observed that najanalgesin, a novel peptide isolated from the venom of Naja naja atra, exerts significant analgesic effects on acute pain in mice and neuropathic pain in rats. The objective of the present study was to determine whether spinal glia are associated with the antinociceptive effect of najanalgesin in an L5 spinal nerve ligation (SNL) rodent model of neuropathic pain. Mechanical allodynia developed after surgery, and hypersensitivity was significantly attenuated by the intrathecal administration of najanalgesin. The inhibitory effect of najanalgesin was significantly (p<0.05) enhanced after pretreatment with fluorocitrate (a glial cell antagonist). In addition, the astrocyte activation was attenuated following najanalgesin treatment in the dorsal horn of neuropathic rats, as assessed by immunohistology and Western blotting. The tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) content of cerebral spinal fluid and cell culture supernatants changed significantly after najanalgesin administration. The results suggest that najanalgesin may exert its anti-allodynic effect by altering astrocyte cell function.