AbstractAimsThe clinical use of brain stimulation is attractive for patients who have side effects or tolerance. However, studies on insular cortex (IC) stimulation are lacking in neuropathic pain. The present study aimed to investigate the effects of IC stimulation (ICS) on neuropathic pain and to determine how ICS modulates pain.MethodsChanges in pain behaviors were observed following ICS with various parameters in neuropathic rats. Western blotting was performed to assess molecular changes in the expression levels of phosphorylated extracellular signal‐regulated kinase (pERK), neurons, astrocytes, and microglia between experimental groups. Immunohistochemistry was performed to investigate the colocalization of pERK with different cell types.ResultsThe most effective pain‐relieving effect was induced at 50 Hz–120 μA in single trial of ICS and it maintained 4 days longer after the termination of repetitive ICS. The expression levels of pERK, astrocytes, and microglia were increased in neuropathic rats. However, after ICS, the expression levels of pERK were decreased, and colocalization of pERK and neurons was reduced in layers 2–3 of the IC.ConclusionThese results indicated that ICS attenuated neuropathic pain by the regulation of pERK in neurons located in layers 2–3 of the IC. This preclinical study may enhance the potential use of ICS and identify the therapeutic mechanisms of ICS in neuropathic pain.