Recent clinical trials report that metformin, an activator of AMP-activated protein kinase (AMPK) used to treat type 2 diabetes, significantly reduces the risk of stroke by actions that are independent of its glucose-lowering effects. However, the underlying molecular mechanisms are not known. Here, we tested the possibility that acute metformin preconditioning confers neuroprotection by pre-activation of AMPK-dependent autophagy in a rat model of permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats were pretreated with either vehicle, an AMPK inhibitor, Compound C, or an autophagy inhibitor, 3-methyladenine, and were injected with a single dose of metformin (10 mg kg(-1), i.p.). Then, AMPK activity and autophagy biomarkers in the brain were assessed. At 24 h after metformin treatment, rats were subjected to pMCAO; infarct volume, neurological deficits and cell apoptosis were evaluated 24 and 96 h later. A single dose of metformin significantly activated AMPK and induced autophagy in the brain. The enhanced autophagic activity was inhibited by Compound C pretreatment. Furthermore, acute metformin preconditioning significantly reduced infarct volume, neurological deficits and cell apoptosis during a subsequent focal cerebral ischaemia. The neuroprotection mediated by metformin preconditioning was fully abolished by Compound C and partially inhibited by 3-methyladenine. These results provide the first evidence that acute metformin preconditioning induces autophagy by activation of brain AMPK, which confers neuroprotection against subsequent cerebral ischaemia. This suggests that metformin, a well-known hypoglycaemic drug, may have a practical clinical use for stroke prevention. © 2014 The British Pharmacological Society.