<p>Electroacupuncture (EA) has emerged as a promising alternative therapy for ischemic brain injury, yet its underlying molecular mechanisms remain incompletely understood. This study investigated the neuroprotective effects and epigenetic mechanisms of EA in a mouse model of ischemic stroke. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in mice. EA was applied at Baihui (GV20) and left Zusanli (ST36) acupoints following surgery. Neurological deficits were assessed, infarct volume was measured by TTC staining, and neuronal damage was evaluated by Nissl staining. RhoGDIα protein expression was determined by Western blot. Global DNA methylation was quantified by ELISA, and promoter-specific methylation of RhoGDIα was examined by quantitative methylation-specific PCR (MSP), and mRNA expression of candidate DNA methyltransferases (DNMTs) and demethylases (TETs) was measured by qPCR. EA alleviated MCAO-induced brain injury, as reflected by improved neurological scores, reduced infarct volume, and attenuated neuronal loss. RhoGDIα expression was downregulated after MCAO and restored by EA treatment. Pharmacological inhibition of RhoGDIα abolished the neuroprotective effects of EA, underscoring its essential role in EA-mediated protection. Mechanistically, MCAO induced hypermethylation of the RhoGDIα promoter, which was reversed by EA, leading to restoration of RhoGDIα expression. Furthermore, co-administration of the DNA methylation inhibitor 5-Azacytidine synergistically enhanced the neuroprotective efficacy of EA. Our findings demonstrate that EA ameliorates ischemic brain injury in association with epigenetic regulation of RhoGDIα via promoter demethylation. This study identifies RhoGDIα as a key mediator of EA-induced neuroprotection and suggests that the DNA methylation–RhoGDIα axis may represent a promising therapeutic target for ischemic stroke.</p>