Ischemic stroke is one of the leading causes of death globally, and inflammation is considered as a vital contributor to the pathophysiology of ischemic stroke. Recently, microRNA-421-3p-derived macrophages is found to promote motor function recovery in spinal cord injury. Here, we explored whether microRNA-421-3p is involved in inflammation responses during cerebral ischemia/reperfusion (I/R) injury and its molecular mechanism. An in vivo experimental animal model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro model of microglial subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) were used. The effects of microRNA-421-3p on cerebral I/R injury and its underlying mechanism were detected by quantitative real-time PCR, western blotting, immunofluorescence staining, RNA immunoprecipitation, flow cytometry, luciferase reporter assay, and bioinformatics analysis. We find that microRNA-421-3p is significantly decreased in cerebral I/R injury in vitro and in vivo. Furthermore, overexpression of microRNA-421-3p evidently suppresses pro-inflammatory factor expressions and inhibits NF-κB p65 protein expression and nuclear translocation in BV2 microglia cells treated with OGD/R. However, microRNA-421-3p neither promotes p65 mRNA expression, nor affects p65 mRNA or protein stability. Moreover, we find the m6A 'reader' protein YTH domain family protein 1 (YTHDF1) is the specific target of microRNA-421-3p, and YTHDF1 specifically binds to the m6a site of p65 mRNA to promote its translation. microRNA-421-3p prevents inflammatory response in cerebral ischemia/reperfusion injury through targeting YTHDF1 to inhibit p65 mRNA translation. These findings provide novel insights into understanding the molecular pathogenesis of cerebral I/R injury. Copyright © 2020 Elsevier B.V. All rights reserved.