To explore the mechanism of TLR4/NF-κB regulation of microglia-mediated inflammation after NSC-Exos transplantation. In this study, a right middle cerebral artery occlusion model (MCAO) was constructed in SD rats, NSC-Exos was injected into the lateral ventricle through brain stereo localization. TAK-242 was given 1 day before MCAO in rats. Histopathological changes, microglia-mediated inflammatory factor, colocalized number of CD86/Iba1 and CD206/Iba1 cells, and TLR4/NF-κB were detected in rats. After brain injury, the number of CD86/Iba1 cells increased, and the expression of pro-inflammatory factors, TLR4 and NF-κB were increased. When treatment with NSC-Exos, the number of CD206/Iba1 cells were increased, and the expression of anti-inflammatory factors, TLR4 and NF-κB were decreased, promoting anti-inflammatory phenotype polarization. More importantly, TAK-242 reversed the effect of NSC-Exos transplantation in animal model. These results suggest that NSC-Exos can improve the microglia-mediated inflammatory response in rats with ischemic brain injury, which may be related to the regulation of TLR4/NF-κB pathway.