The reperfusion after an acute ischemic stroke can lead to a secondary injury, which is ischemia-reperfusion (I/R) injury. During ischemia, the reactive oxygen species (ROS) is over-produced, mostly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Besides, miRNAs are also associated with neuronal death in ischemic stroke. MiR-124-5p is selectively expressed within central nervous system (CNS) and is predicted to bind to NOX2 directly. Herein, we successfully set up cerebral I/R injury model in rats through middle cerebral artery occlusion (MCAO) surgery. After 12 h or 24 h of refusion, the superoxide dismutase (SOD) activity was significantly inhibited, accompanied by NOX2 protein increase within MCAO rat infarct area. In vitro, oxygen-glucose deprivation/refusion (OGD/R) stimulation on PC-12 cells significantly increased NOX2 protein levels, ROS production, and the cell apoptosis, while a significant suppression on SOD activity; OGD/R stimulation-induced changes in PC-12 cells described above could be significantly attenuated by NOX2 silence. In vivo, miR-124 overexpression improved, whereas miR-124 inhibition aggravated I/R injury in MCAO rats. miR-124-5p directly bound to the CYBB 3'-untranslated region (UTR) to negatively regulate CYBB expression and NOX2 protein level. In vitro, miR-124 overexpression improved, while NOX2 overexpression aggravated OGD/R-induced cellular injuries; NOX2 overexpression significantly attenuated the effects of miR-124 overexpression. Besides, miR-124 overexpression significantly repressed NF-κB signaling activation and TNFα and IL-6 production through regulating NOX2. In conclusion, miR-124-5p/NOX2 axis modulates NOX-mediated ROS production, the inflammatory microenvironment, subsequently the apoptosis of neurons, finally affecting the cerebral I/R injury.