Endothelial tight junction is a crucial intracellular junctional structure that controls paracellular permeability across vascular endothelium. Oxidative stress-mediated elevation in endothelial permeability is associated with pathogenesis of several cardiovascular diseases. In the present research, the regulation of farrerol on occludin, a transmembrane proteins associated with endothelial tight junction, was investigated in hydrogen peroxide-induced human endothelium-derived EA.hy926 cells. Western blot analysis demonstrated that H2O2 exposure caused a significant decrease in occludin expression, but had little effect on ZO-1 expression, and the decrease of occludin expression was significantly attenuated by farrerol in a dose-dependent manner. Meanwhile, immunofluorescent staining assay also demonstrated that the loss of occludin expression induced by H2O2 exposure was restored by farrerol pretreatment. Further investigations showed that farrerol prevented H2O2-induced activation of extracellular signal-regulated kinase (ERK) 1/2 in a dose-dependent manner. The use of U0126, a specific inhibitor of MEK1/2, proved that H2O2-induced decrease of occludin in EA.hy926 cells was likely associated with activation of ERK1/2, which indicated that the regulation of farrerol on occludin expression in H2O2-induced EA.hy926 cells was likely related to the modulation of ERK1/2 activation. In conclusion, the present study demonstrates for the first time that farrerol has potential effects on oxidative stress-induced endothelial tight junction disruption and suggests that farrerol is a potential candidate for the intervention of endothelial permeability-associated cardiovascular diseases.