Complement component C3 (C3) plays a central role in microglial neurotoxicity following cerebral ischemia/reperfusion (I/R) injury. In this study, we focused on the role of nanoparticles loaded with C3 siRNA (NP) in inhibiting microglial neurotoxicity after brain (I/R) injury. NP inhibited the hypoxia/re-oxygenation-induced increase in C3 expression in microglia in vitro. Importantly, treatment with NP decreased C3b deposition on neurons and reduced microglia-mediated neuronal damage under hypoxia/re-oxygen conditions. Nanoparticles could effectively deliver C3-siRNA from the blood into ischemic penumbra across the blood-brain barrier (BBB) and significantly decrease C3 expression in microglia and ischemic brain tissue, while reducing the number of infiltrating inflammatory cells and the concentration of pro-inflammatory factors in the penumbra. Furthermore, NP also prevented neuronal apoptosis, reduced the volume of the ischemic zone, and substantially improved functional recovery after I/R injury. Therefore, the NP-induced inhibition of microglial neurotoxicity represents a novel therapeutic strategy for treating brain I/R injury.