The role of endothelial cells in acute lung injury (ALI) has been widely elaborated, but little is known about the role of different subtypes of endothelial cells in ALI. ALI models were established by lipopolysaccharide. Single-cell RNA sequencing was used to identify differential molecules in endothelial subtypes and the heterogeneity of lung immune cells. Specific antibodies were used to block insulin-like growth factor binding protein 7 (IGFBP7), and AAV was used to specifically knock down IGFBP7. Here, we found that IGFBP7 was the most differentially expressed molecule in diverse subsets of endothelial cells and that IGFBP7 was strongly associated with inflammatory responses. Elevated IGFBP7 significantly exacerbated barrier dysfunction in ALI, whereas blockade of IGFBP7 partially reversed barrier damage. General capillary cells are the primary source of elevated serum IGFBP7 after ALI. Using single-cell RNA sequencing, we identified significantly increased Clec4n neutrophils in mice with ALI, whereas IGFBP7 knockdown significantly reduced infiltration of Clec4n cells and mitigated barrier dysfunction in ALI. In addition, we found that IGFBP7 activated the NF-κB signaling axis by promoting phosphorylation and ubiquitination degradation of F-box/WD repeat-containing protein 2 (FBXW2), thereby exacerbating barrier dysfunction in ALI. Taken together, our data indicate that blockade of serum IGFBP7 or IGFBP7 depletion in general capillary cells reversed barrier damage in ALI. Therefore, targeting IGFBP7 depletion could be a novel strategy for treating ALI.