The purpose of this study was to explore the therapeutic time window and mechanism of tetramethylpyrazine on transient focal cerebral ischemia/reperfusion injury. Middle cerebral artery occlusion was conducted in male Sprague-Dawley rats and 20mg/kg tetramethylpyrazine was injected intraperitoneally at different time points. Neurological deficit scores and brain infarction volumes were measured 72 h after reperfusion started. The expression of thioredoxin and thioredoxin reductase were examined at 6h and at 24h after reperfusion. Our results included the findings of a significant reduction in neurological deficit scores and infarction volume in the treatment group as compared to the control group. Ischemia/reperfusion injury resulted in a decrease in the expression of thioredoxin, while tetramethylpyrazine administration greatly elevated the expression of thioredoxin-1/thioredoxin-2 mRNA and thioredoxin reductase-1/thioredoxin reductase-2 mRNA. These findings suggest that administration of tetramethylpyrazine, within a 4h time period post-transient focal stroke, may reduce cerebral ischemic reperfusion damage. Moreover, the neuroprotective effect of tetramethylpyrazine may be mediated, in part, by an increase in genetic transcription of thioredoxin.