Accumulating evidence suggests that ischemic preconditioning (IPC) increases cerebral tolerance to the subsequent ischemic exposure. However, the underlying mechanisms are still not fully understood. In the present study, we tested the hypothesis that AMP-activated protein kinase (AMPK)-dependent autophagy contributed to the neuroprotection of IPC in rats with permanent cerebral ischemia. Male Sprague-Dawley rats were pretreated with vehicle, compound C (an AMPK inhibitor), or 3-methyladenine (3-MA, an autophagy inhibitor) and then were subjected to IPC induced by a 10-min middle cerebral artery occlusion. Afterward, the brain AMPK activity and autophagy biomarkers were measured. At 24 h after IPC, permanent cerebral ischemia was induced in these rats, and infarct volume, neurological deficits as well as cell apoptosis were evaluated 24 h later. We demonstrated that IPC activated AMPK and induced autophagy in the brain, which was accompanied by a reduction of infract volume, neurological deficits, and cell apoptosis after cerebral ischemia. Meanwhile, the IPC-induced autophagy was inhibited by compound C while the neuroprotection of IPC was abolished by compound C or 3-MA. These findings suggest that AMPK-mediated autophagy contributes to the neuroprotection of IPC, highlighting AMPK as a therapeutic target for stroke prevention and treatment.