Erythropoietin (EPO) is not limited to hematopoiesis, it may act as a protective cytokine. In this research, retinal pigment epithelial (RPE) cell viability, cell monolayer integrity, RPE barrier permeability, distribution of the junction proteins ZO-1, occludin and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were monitored to evaluate the effect of EPO on non-lethal H2O2-induced RPE barrier hyperpermeability. Results showed that EPO increased the viability of H2O2-treated RPE cells, the disruption of junction proteins and the higher permeability caused byH2O2 waspartially prevented by EPO pre-treatment. EPO treatment also induced lower MDA levels and higher SOD activity in H2O2 treated RPE cells. So it is concluded that non-lethal concentrations of H2O2 could damage RPE barrier and destroy its integrity. EPO showed the protective effects on H2O2-induced hyperpermeability by stabilizing the distribution of junction proteins and reducing oxidative stress. These results indicated that EPO may have the therapeutic potential use in treatment of eye diseases involving RPE barrier hyperpermeability induced by oxidative stress.