Several studies demonstrate that hemolysis and free heme in circulation causes endothelial barrier dysfunction and is associated with severe pathological conditions like acute respiratory distress syndrome, acute chest syndrome, and sepsis. Yet, the precise molecular mechanisms involved in the pathology of heme induced barrier disruption still remains to be elucidated. In this study, we investigated the role of free heme on the endothelial barrier integrity and the mechanisms of heme-mediated intracellular signaling in human lung microvascular endothelial cells (HLMVEC). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity in HLMVEC. An investigation into barrier proteins revealed that heme primarily affects the tight junction proteins, zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of the endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in the HLMVEC and in mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we show that this KO effectively decreased heme induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.