Recent evidences have implicated that SENP3 is a deSUMOylase which possesses neuronal damage effects in cerebral ischemia. However, its role in microglia remains poorly understood. Here, we found that SENP3 was upregulated in the peri-infarct areas of mice following ischemic stroke. Furthermore, knockdown of SENP3 significantly inhibits the expression of proinflammatory cytokines and chemokines in microglial cells. Mechanistically, SENP3 can bind and then mediated the deSUMOylation of c-Jun, which activated its transcriptional activity, ultimately followed by the activation of MAPK/AP-1 signaling pathway. In addition, microglia-specific SENP3 knockdown alleviated ischemia-induced neuronal damage, and markedly diminished infract volume, ameliorated sensorimotor and cognitive function in animals subjected to ischemic stroke. These results indicated SENP3 functions as a novel regulator of microglia-induced neuroinflammation by activating the MAPK/AP-1 signaling pathway via mediating the deSUMOylation of c-Jun. Interventions of SENP3 expression or its interaction with c-Jun would be a new and promising therapeutic strategy for ischemic stroke.© 2023 The Author(s).