Background: Cerebral infarction ranks as the second leading cause of disability and death globally, and inflammatory response of glial cells is the main cause of brain damage during cerebral infarction. Methods: Studies have shown that mesenchymal stem cells (MSCs) can secrete exosomes and contribute to cerebral disease. Here, we would explore the function of MSC-derived exosome in cerebral infarction. Results: Microarray indicated a decrease of miR-542-3p and an increase of Toll-Like Receptor 4 (TLR4) in middle cerebral artery occlusion (MCAO) mice comparing with sham mice. And luciferase and RIP analysis indicated a binding of miR-542-3p and TLR4. Then, we injected AAV9-miR-542-3p into paracele of sham or MCAO mice. Functional analysis showed that AAV9-miR-542-3p inhibited infarction area and the number of degenerating neurons and suppressed inflammatory factors’ expression and inflammatory cell infiltration. As well, transfection of miR-542-3p mimics into HA1800 cells underwent oxygen and glucose deprivation (OGD). Similarly, overexpression of miR-542-3p alleviated OGD induced cell apoptosis, ROS, and activation of inflammation response. Moreover, miR-542-3p could be packaged into MSCs and secreted into HA1800 cells. The extractive exosome-miR-21-3p treatment relieved MCAO- or OGD-induced cerebral injury and inflammation through targeting TLR4. Conclusion: These results confirmed that MSC-derived exosome miR-542-3p prevented ischemia-induced glial cell inflammatory response via inhibiting TLR4. These results suggest possible therapeutic strategies for using exosome delivery of miR-542-3p to cure cerebral ischemic injury. © 2021, The Author(s).