Abstract: The pathology of ischemic stroke and the mechanism of thrombolytic therapy induced lethal hemorrhagic transformation (HT) involve disruption of the blood brain barrier (BBB) . This study evaluated the effect of regulatory T cells (Tregs) transfer on ischemic stroke and rtPA-enhanced HT. In addition, we further investigated the mechanism underlying Treg-afforded BBB protection. Cerebral ischemia was induced in mouse by suture middle cerebral artery occlusion (MCAO) for 1h, or for 2h followed by intravenous rtPA infusion leading to HT. Systemic administration of purified Tregs at 2, 6, or 24 hours after the surgery, resulted in marked reduction of brain infarct and rtPA-induced cerebral hemorrhage. Treg-afforded neuroprotection was accompanied by attenuated blood–brain barrier (BBB) disruption during early stages of ischemia in these two different model types. Further studies suggested that Tregs inhibited the elevation of matrix metallopeptidase-9 (MMP9) and chemokine (C-C motif) ligand 2 (CCL2) expression after stroke , thus preventing proteolytic damage of the BBB. Using MMP9 knockout and CCL2 knockout mice, we found that both molecules partially contribute to the protective actions of Tregs. Using In vitro model of BBB, we confirmed that Tregs inhibited tPA-induced endothelial expression of CCL2 and protected BBB against ischemic challenge. Clinical data revealed a significant decrease in the number of circulating Tregs upon stroke onset. The prolonged loss of circulating Tregs is correlated with poor stroke outcomes. It is concluded that Tregs reduces ischemia and rtPA-induced BBB damage by two inhibitory mechanisms targeting both CCL2 and MMP9. Tregs may represent a potent cell-based therapy to decrease the brain infarct and increase the safety of thrombolytic treatment for stroke.