MicroRNA-181c (miR-181c) has been reported to be highly expressed in the brain, but downregulated in acute ischemic stroke patients. However, the underlying mechanism of miR-181c in regulating cerebral ischemic injury (I/R) remains poorly understood. The aim of this study was to explore the molecular basis of miR-181c in regulating cerebral I/R. It was found that the overexpression of miR-181c mediated by recombinant adeno-associated virus (AAV) vector infection significantly promoted neuron death induced by oxygen-glucose deprivation (OGD)/ reoxygenation in hippocampal neuron, whereas the inhibition of miR-181c provided protective effects against OGD/reoxygenation-induced cell death. In addition, c-Fos expression was decreased and increased though overexpression or inhibition of miR-181c. c-Fos was further determined to a putative target of miR-181c by dual-luciferase reporter assay. miR-181c overexpression also inhibited the expression of the downstream gene of c-Fos, including AP-1 and NFATc1, whereas the inhibition of miR-181c upregulated the expression of AP-1 and NFATc1 in neurons after OGD/reoxygenation. Interestingly, c-Fos siRNA apparently abolished the protective effect of anti-miR-181c on OGD/reoxygenation-induced cell death. These observations determine that downregulated miR-181c ameliorates I/R via increasing the expression of c-Fos and its downstream genes, which will provide a new and promising therapeutic target for cerebral I/R.