Gamma-linolenic acid (GLA) is found in animals and plants that play a role in brain function and metabolism. This study aimed to investigate the analgesic effects of GLA on peripheral formalin injection. Wistar rats were randomly assigned to four groups: Sham, formalin, formalin/GLA 100mg/kg, and formalin/GLA 150mg/kg. The Formalin test was utilized to create a pain model. A tissue sample was prepared from the spinal cords of rats to measure oxidative stress parameters and pro-inflammatory cytokines. Furthermore, the authors analyzed the expression of c-Fos protein in the spinal cords. Our findings demonstrate that GLA has a reliable pain-relieving effect in the formalin test. GLA 100 increased superoxide dismutase (SOD) (P<0.05), glutathione (GSH) (P<0.001), and catalase (CAT) (P<0.05), and decreased the levels of c-Fos (P<0.001), interleukin-1 beta (IL-1β) (P<0.001), tumour necrosis factor-alpha (TNF-α) (P<0.001), and malondialdehyde (MDA) (P<0.001) in the spinal cord. Also GLA 150 increased SOD (P<0.05), GSH (P<0.001), and CAT (P<0.05) and decreased the levels of c-Fos (P<0.001), IL-1β (P<0.001), TNF-α (P<0.001), and MDA (P<0.001) in the spinal cord. The findings have validated the antinociceptive impact of GLA and hinted towards its immunomodulatory influence in the formalin test.