Background The incidence, mortality and disability rates of ischemic stroke are high, and the reperfusion injury after thrombolytic therapy has a great impact on patients. Acupuncture is a characteristic therapy for the treatment of the disease, but the action mechanism remains unclear. Objective To observe the effect of Tongdu Tiaoshen electroacupuncture (EA) pretreatment on miR-124-3p/glycogen synthase kinase β (GSK-3β) /cyclin D (Cyp-D) signaling pathway and mitochondrial permeability transition pore (MPTP) of cerebral ischemia-reperfusion injury (CIRI) rats, and explore its possible mechanism of prevention and control of CIRI. Methods From June to August 2022, a total of 100 clean SD rats were randomly divided into the sham operation group, model group, EA group, agonist group and EA + inhibitor group, with 20 rats in each group. For 7 d of intervention before modeling, in the EA group and EA + inhibitor group, "Baihui" (GV 20) , "Fengfu" (GV 16) and "Dazhui" (GV 14) were selected to perform electroacupuncture 1 time a day for 7 days. For 24 h before modeling, miR-124 agonist and inhibitor (5 nmol) were injected into the lateral ventricles in the EA + agonist group and inhibitor group. Except for the sham operation group, the right cerebral ischemia-reperfusion model of rats was prepared by the modified suture method in the rest groups. The right cerebral cortex of rats was taken, the degree of neurological impairment in each group was observed using mNSS scale and TTC staining, the nerve cell injury was observed by TUNEL staining and transmission electron microscopy. The expressions of GSK-3β, p-GSK-3β, Cyp-D, Cyt-C and Caspase-3 in cerebral cortex of each group were detected by immunofluorescence staining, Western blot and real-time quantitative PCR. The degree of MPTP openness was detected by flow cytometry, the higher rate of MPTP-positive cells indicated greater degress of MPTP openness. Results Except for the sham operation group, all rest groups of rats were successfully modeled. Compared with the sham operation group, mNSS score and infarct volume of brain tissue were increased, mitochondrial structure was seriously damaged, cell apoptosis index was increased, p-GSK-3β positive expression was reduced, Cyp-D positive expression was enhanced, miR-124-3p, Cyp-D, Cyt-C and Caspase-3 mRNA expressions were increased, p-GSK-3β/ GSK-3β ratio was decreased, relative Cyp-D, Cyt-C, and Caspase-3 protein expressions were increased, and MPTP openness degree was increased in the model group (P