Background <p>Electroacupuncture is widely accepted to treat pain related conditions, but detailed mechanisms remain unknown.</p> Objective <p>To explore cortical and subcortical subnuclei involved in electroacupuncture stimulation (EAS) analgesia by observing EAS’s analgesic efficacy and c-fos expression changes, providing a basis for neural circuit research and clinical transcranial magnetic stimulation (TMS) therapy.</p> Design, setting, participants and interventions <p>A chronic inflammatory pain model was established using knee osteoarthritis (KOA). Bilateral Zusanli was selected for electroacupuncture intervention. Von Frey test, open field test, elevated plus maze, and tail suspension test, and immunohistochemical staining were performed.</p> Main outcomes measures <p>Changes in mechanical pain threshold and pain-related emotional behaviors and distribution of c-fos positive cells in cortical and subcortical nuclei.</p> Results <p>Electroacupuncture significantly increased mechanical pain thresholds in KOA model mice. KOA modeling caused c-fos downregulation in the motor cortex, insular cortex, secondary auditory cortex, dorsal peduncular cortex, temporal association cortex, caudate putamen, lateral septal nucleus, accumbens nucleus, and the anterior cortical amygdaloid area. Electroacupuncture at Zusanli reversed these changes, upregulating c-fos in abovementioned brain regions, and additionally upregulated c-fos expression in the granular insular cortex, extended amydala.</p> Conclusion <p>Inflammatory pain induces widespread inhibition of neuronal activity in cortical and subcortical nuclei. The core mechanisms of electroacupuncture analgesia may involve direct reversal of abnormal inhibition in the lateral septal nucleus, caudate putamen, accumbens nucleus, and the anterior cortical amygdaloid area and activation of the granular insular cortex, medial septal nucleus and the extended amygdala for pain information integration.</p>