Exogenously administered recombinant human erythropoietin (rhEPO) has been reported to exhibit neuroprotective effects in animal models. However, there are still have some controversies that combination of EPO and tissue plasminogen activator (tPA) in acute ischemic stroke. In the present study, we investigated the effects of local intra-arterial infusion of low-dose EPO in combination with tPA on focal cerebral ischemic stroke. Sixty adult male Sprague-Dawley rats were randomly divided into five groups, including sham, vehicle, EPO, tPA, and EPO+tPA groups. Rats were subjected to middle cerebral artery occlusion (MCAO) and administrated with EPO (800 U/kg, middle cerebral artery injection), tPA (10 mg/kg, tail vein injection), EPO+tPA, or saline (vehicle) onset of reperfusion. Neurobehavioral deficits, infarct volume, brain edema, the expression of tight junction proteins (Claudin-5, Occludin), and AQP4 were assessed following 2 h ischemia and 24 h reperfusion. The number of apoptotic cells in the periinfarct region was detected by the terminal deoxyribonucleotide transferase dUTP nick end labeling (TUNEL) staining. The neurobehavioral deficits, brain infarct volume, edema volume, TUNEL-positive cells and downregulation of Claudin-5 and Occludin were alleviated by EPO or EPO plus tPA, following the ischemia/reperfusion (I/R) in rats. The EPO and EPO plus tPA both reduced the upregulation of AQP4 in the ischemic brain tissue. Our data demonstrate local intra-arterial infusion of low-dose EPO in combination with tPA protected against focal cerebral ischemia in rats manifested by a decrease in brain edema and blood-brain barrier (BBB) disruption after 2 h ischemia and 24 h reperfusion.