The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung micro-vascular endothelial cells exposed to IH consisting alternating cycles of 1.5% O₂ for 30s followed by 20% O₂ for 5 min. IH decreased trans-endothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus-dependent and reversible after re-oxygenation. IH exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins and zona occludens-1, junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal regulated kinase (ERK) and c-jun NH₂-terminal kinase (JNK) were phosphorylated in IH exposed cells. Inhibiting either ERK or JNK prevented IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane permeable anti-oxidant prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to re-organization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.