Acute respiratory distress syndrome (ARDS) is an acute inflammatory lung injury for which effective therapeutic agents are lacking. Excessive endothelial cell (EC) activation is a critical trigger of inflammation. Extracellular vesicles (EVs) are increasingly recognized as prominent regulators of inflammatory responses. The previous study identified secretory autophagosomes (SAPs), a novel class of EVs, as a prognostic marker in ARDS, raising questions of whether and how they are involved in the pathogenesis of ARDS. Here, it is shown that inflamed macrophage-derived SAPs (MSAPs) exacerbate lung injury by weakening the role of ECs as gatekeepers of immune cell transport within the lung. Bioinformatics and functional studies reveal that tRF-5004b is a key molecule of MSAPs in mediating endothelial activation. Mechanically, tRF-5004b directly interacts with the nuclear transporter KPNA2, thereby facilitating the association between KPNA2 and the transcription factor p65. This interaction enhances p65 nuclear translocation, a process implicated in EC activation. Additionally, the level of tRF-5004b is positively correlated with the severity of ARDS, and patients with high tRF-5004b levels have a poor prognosis. Overall, it is found that tRF-5004b-enriched SAPs induce acute lung injury by promoting p65 nuclear translocation to activate ECs, suggesting that tRF-5004b may be a novel therapeutic target for ARDS.