Abstract Cerebral ischemic stroke is a leading cause of morbidity and mortality globally. However, the mechanisms underlying ischemic stroke injury remain poorly understood. Here, it is found that deficiency of the ubiquitin‐specific protease USP25 significantly aggravate ischemic stroke injury in mice. USP25 has no impact on neuronal death under hypoxic conditions, but reduced ischemic stroke‐induced neuronal loss and neurological deficits by inhibiting microglia‐mediated neuroinflammation. Mechanistically, USP25 restricts the activation of NF‐κB and MAPK signaling by regulating TAB2. As a deubiquitinating enzyme, USP25 removeds K63‐specific polyubiquitin chains from TAB2. AAV9‐mediated TAB2 knockdown ameliorates ischemic stroke injury and abolishes the effect of USP25 deletion. In both mouse and human brains, USP25 is markedly upregulated in microglia in the ischemic penumbra, implying a clinical relevance of USP25 in ischemic stroke. Collectively, USP25 is identified as a critical inhibitor of ischemic stroke injury and this data suggest USP25 may serve as a therapeutic target for ischemic stroke.