T-LAK-cell-originated protein kinase (TOPK), a MAPKK-like kinase, is crucial for neural progenitor cell proliferation; however, the function of TOPK and the molecular mechanism underlying cerebral ischemia-reperfusion injury remains unknown. Therefore, we investigated the role of TOPK in experimental stroke. Sprague-Dawley rats underwent transient middle cerebral artery occlusion (tMCAO) and reperfusion, and TOPK small interfering RNA (siRNA) was delivered by intracerebroventricular injection at the beginning of MCAO. After TOPK overexpression and H2O2 stimulation in PC12 neuronal cells, antioxidative proteins, apoptosis-related proteins and signal pathways were detected by western blot analysis, the levels of the peroxidation products (malondialdehyde and 3-nitrotyrosine) were measured with ELISA. Phosphorylation of TOPK was increased in rat cortical neurons following tMCAO. TOPK overexpression in PC12 cells augmented levels of antioxidative proteins (peroxiredoxin 1 and 2, heme oxygenase 1 and manganese superoxide dismutase), as well as total superoxide dismutase activity, along with inhibition of malondialdehyde and 3-nitrotyrosine upon H2O2 stimulation. TOPK overexpression increased cell viability and reduced expression of caspase 3 and caspase 12 in PC12 cells in response to H2O2. The p-ERK level was increased by TOPK overexpression, and antioxidative protection afforded by TOPK was abolished by blocking the extracellular signal-regulated kinase pathway in PC12 cells. TOPK siRNA increased the infarct volume and reduced total superoxide dismutase activity in the cortex in vivo after MCAO. These data reveal that activating TOPK confers neuroprotection against focal cerebral ischemia-reperfusion injury by antioxidative function, in part through activation of the extracellular signal-regulated kinase pathway.