Administration of recombinant human erythropoietin (rhEPO) protects neurons from injury after brain ischemia-reperfusion (I/R), which is in part mediated by ameliorating the blood-brain barrier (BBB) leakage. But the mechanism of rhEPO's protective effects on BBB remains unclear. This study aims to investigate the effects of rhEPO on BBB integrity and the expressions of tight junctions (TJs) associated proteins of zonula occluden-1 (ZO-1), occludin, and claudin-5 in cerebral I/R rats. These rats underwent 2 h of ischemia and then were reperfused for up to 3 and 72 h. Animals were randomly divided into five groups: sham-operated group, I/R 3 h and I/R 72 h group (2 ml saline was injected intraperitoneally just before the onset of ischemia), rhEPO + I/R 3 h, and rhEPO + I/R 72 h group (5,000 U/kg rhEPO diluted in 2 ml saline solution was injected intraperitoneally just before the onset of ischemia). We verified that rhEPO could decrease the BBB leakage induced by I/R injury detected by Evans blue extravasation. 2, 3, 5-Triphenyltetrazolium chloride staining results showed that rhEPO decreased infarct volume after cerebral I/R. TJ integrity was partly restored by rhEPO observed by transmission electron microscopy. The mRNA and protein expression levels of ZO-1, occludin, and claudin-5 were significantly increased compared with I/R groups at the same reperfusion time point by reverse transcriptase-polymerase chain reaction and Western blot assays. The treatment of rhEPO induced the redistribution of ZO-1, occludin, and claudin-5 in cerebral microvessels observed by immunohistochemical staining. Compared with I/R groups, the mRNA level of tumor necrosis factor-alpha (TNF-alpha) in cerebral microvessels decreased markedly after rhEPO treatment, accompanied with reduced TNF-alpha protein level and nuclear factor-(DB)-B-0 (NF-(DB)-B-0) p65 activation detected by enzyme-linked immunosorbent assay. These results suggested that the protective mechanism of rhEPO on BBB after cerebral I/R injury was associated with the upregulation of TJ-associated proteins. The downregulated TNF-alpha levels and NF-(DB)-B-0 activation induced by rhEPO might be involved in this process.