Cerebral ischemia and reperfusion increase superoxide anions (O(2)(*-)) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O(2)(*-) overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early postreperfusion periods after tFCI, STAT3 was rapidly downregulated, and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O(2)(*-) in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.