Abstract: Objective To investigate nestin activation in rat brain subjected to ischemia-reperfusion injury and its changes in response to Tongxinluo treatment. Methods Cerebral ischemia was induced by temporary middle cerebral artery occlusion (MCAO) in rats. At 3, 7, 14 and 21 days after MCAO, nestin expression in the ependyma, subventricular zone (SVZ), hippocampal subdentate gyrus zone (HDG) of the rats treated with Tongxinluo were guantified by immunohistochemistry. Results Compared with the sham operation group, nestin was significantly increased 7, 14 and 21 days after MCAO (P<0.05), and immunofluorescence of BrdU+nestin-positive neurons significantly increased in the SVZ. After treatment with Tongxinluo, the number of BrdU-positive neurons and BrdU+nestin-positive neurons significantly increased as compared with MCAO group (P<0.05). Conclusion Focal cerebral ischemia in the rat results in rapid response and proliferation of neural stem cells in the SVZ and HDG in the ischemic hemisphere, and Tongxinluo may enhance the differentiation and proliferation capacity of the neural stem cells after MCAO.