Background Preconditioning with repeated electroacupuncture (EA) could mimic ischemic preconditioning to induce cerebral ischemic tolerance in rats. The present study was designed to investigate whether mu(μ)-, delta(δ)- or kappa(κ)-opioid receptors are involved in the neuroprotection induced by repeated EA preconditioning.Methods The rats were pretreated with naltrindole (NTI), nor-binaltorphimine (nor-BNI) or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), which is a highly selective δ-, κ- or μ-opioid receptor antagonist respectively, before each EA preconditioning (30 minutes per day, 5 days). Twenty-four hours after the last EA treatment, the middle cerebral artery occlusion (MCAO) was induced for 120 minutes. The brain infarct volume was determined with 2,3,5-triphenyltetrazolium chloride staining at 24 hours after MCAO and compared with that in rats which only received EA preconditioning. In another experiment, the met-enkephalin-like immunoreactivity in rat brain was investigated by immunohistochemistry in both EA preconditioning and control rats.Results The EA preconditioning reduced brain infarct volume compared with the control rats (P=0.000). Administration of both NTI and CTOP attenuated the brain infarct volume reduction induced by EA preconditioning, presenting with larger infarct volume than that in the EA preconditioning rats (P＜0.001). But nor-BNI administration did not block the infarct volume reduction induced by EA preconditioning, presenting with smaller infarct volume than the control group rats(P=0.000). The number of met-enkephalin-like immunoreactivity positive neurons in the EA preconditioning rats was more than that of the control rats (P=0.000).Conclusion Repeated EA preconditioning stimulates the release of enkephalins, which may bind δ- and μ-opioid receptors to induce the tolerance against focal cerebral ischemia.