Background: Previous data have reported that Sentrin/SUMO-specific protease 6 (SENP6) is involved in ischaemic brain injury and induces neuronal apoptosis after cerebral ischaemia, but the role of SENP6 in microglia-induced neuroinflammation and its underlying mechanism remain poorly understood. This research systematically explored the function and potential mechanism of SENP6 in microglia-induced neuroinflammation after ischaemic stroke. Results: We first identified an increased protein level of SENP6 in microglia after cerebral ischaemia. Then, we demonstrated that SENP6 promoted detrimental microglial phenotype polarization. Specifically, SENP6-mediated de-SUMOylation of ANXA1 targeted the I kappa B kinase (IKK) complex and selectively inhibited the autophagic degradation of IKK alpha in an NBR1-dependent manner, activating the NF-kappa B pathway and enhancing proinflammatory cytokine expression. In addition, downregulation of SENP6 in microglia effectively reduced cocultured neuronal damage induced by ischaemic stroke. More importantly, we employed an AAV-based technique to specifically knockdown SENP6 in microglia/macrophages, and in vivo experiments showed that SENP6 inhibition in microglia/macrophages notably lessened brain ischaemic infarct size, decreased neurological deficit scores, and ameliorated motor and cognitive function in mice subjected to cerebral ischaemia surgery. Conclusion: We demonstrated a previously unidentified mechanism by which SENP6-mediated ANXA1 de-SUMOylation regulates microglial polarization and our results strongly indicated that in microglia, inhibition of SENP6 may be a crucial beneficial therapeutic strategy for ischaemic stroke.