Objective To investigate the effect of focal cerebral ischemia/ reperfusion injury on TMEM166 and neuron autophagy. Methods In total, fifty C57/ BL6J mice were used to establish the focal cerebral ischemia model through middle cerebral artery occlusion (MCAO) by endovascular suture and were randomly divided into four groups according to different reperfusion time points: 6, 12, 24 and 48 h. Changes in the expression of TMEM166 and the number of LC3?II positive cells in cerebral cortex and basal ganglia of the ischemic hemisphere of the mice were measured by immunochemistry. Protein levels of TMEM166 and LC3?II were detected by Western blotting. Results TMEM166 was induced in mice after cerebral ischemia/ reperfusion injury, peaking at 24 h after injury in the ischemic hemisphere. LC3?II began to increase at 6 h following MCAO, increasing over time to reach a highest level at 24 h after ischemic insult. The changing pattern of TMEM166 was consistent with changes in LC3?II positive cell numbers. No changes of TMEM166 and LC3?II were found in the contralateral hemisphere cerebral cortex and basal ganglia. Conclusions TMEM166 is induced after MCAO, and can lead to neuron autophagy by activating LC3?II.