Background and Purpose 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). Experimental Approach 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. Key Results 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. Conclusions and Implications 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.