Sepsis is a clinical syndrome that reflects an uncontrolled systemic inflammatory response to microbial infections. Endothelial cells, which are highly responsive to their extracellular environment, are the primary targets of sepsis-induced damage. Endothelial-mesenchymal transition (EndMT), characterized by loss of endothelial cell markers and gain of mesenchymal cell markers, contributes to embryonic cardiac formation as well as development of various diseases. We showed that incubation with septic serum induced a rapid loss of endothelial barrier integrity in a human umbilical vein endothelial cell (HUVEC) monolayer. Notably, exposure to septic serum triggered EndMT in HUVECs, companied by increased cell motility and invasion. Furthermore, membrane proteomic analysis was applied to analyze the key mediators in septic serum-induced EndMT. A total of 29 proteins with altered expression level were positively identified. Expression of four proteins with the most significant alteration, including caveolin-1, S100A4, α-enolase and galectin-1, were validated by western blotting. Finally, we showed that restoration of caveolin-1 expression markedly attenuated septic serum-induced EndMT in HUVEC cells. This is the first report showing that endothelial cells undergo EndMT during sepsis. The present study may lead to a better understanding of the biological role of endothelial cells and caveolin-1 during sepsis.