The endothelium is a monolayer of cells which line all of the vasculature where it acts as a barrier to control permeability of water and other molecules out of a blood vessel. Loss of endothelial barrier function resulting in increased endothelial permeability is often seen in pathological inflammation leading to organ damage. The inflammatory cytokine TNF-α activates several different signaling pathways within endothelial cells including the p38 MAPK pathway. TNF-α is known to be elevated in patients with certain inflammatory disorders and is thought to contribute to these disorders by disrupting the integrity of the endothelium leading to increased permeability. Whether TNF-α uses the p38 MAPK pathway to increase permeability remains unknown. The data presented here shows that TNF-α, at least in part, increases permeability by disrupting ZO-1, a key component of the tight junction complex. This data also shows that p38 MAPK activity is not required for the initial increase in permeability (two hours after treatment) caused by TNF-α, but, instead, is essential for the sustained increase in permeability (six hours after treatment). Taken together, our results suggest that targeting the p38 MAPK pathways could provide new therapeutic strategies to block vascular leakage associated with pathological inflammatory conditions and preventing the ensuing organ damage.