Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life-threatening critical syndrome with no effective pharmacotherapy. Extracellular vesicles (EVs) are considered as a new way of long-distance communication between cells. Our previous research using an ex vivo perfused human ALI model suggested that endothelial cell-derived EVs (EC-EVs) mediate the development of ALI/ARDS. However, how EC-EVs aggravate lung injury remains largely unknown. Here we demonstrated that EC-EVs released under inflammatory stimulation can lead to ALI, and the proteomic analysis was performed on EC-EVs to explore the mechanism.