Objective To investigate the effects on acute lung injury (ALI) of CD226 conditional knockout (CD226 CKO) in vascular endothelial cells were investigated in mice with hemorrhagic shock (HS) and its mechanism. Methods Male wild type (WT) and CD226 CKO mice were randomly divided into sham and HS groups: in the sham group, a heart puncture was performed but blood was not drawn; in the HS group, the heart was punctured and 30% of the total blood volume was drawn. To assess lung injury, lung lesions were observed by HE staining. Immunofluorescence histochemical staining was used to detect the expression and distribution of CD31, CD226 in lung tissue and CD3 and CD226 in spleen. In addition, a RNA interering (RNAi) was used to knockdown CD226 in human umbilical vein endothelial cells and a hypoxia model was established. Protein expression of Bcl2 in lung tissue and vascular endothelial cells was detected by Western blotting. Early apoptosis was detected by JC-1 mitochondrial membrane potential staining. Results In the HS groups, CD226 CKO mice showed significantly less ALI than WT mice, and the protein expression of Bcl2 in their lung tissues increased. Furthermore, in vitro cytological models revealed that protein expression of Bcl2 increased and apoptosis decreased in the siCD226 group relative to the siNC group under hypoxia. Conclusion CD226 CKO in vascular endothelial cells reduces ALI in mice with HS, and this effect is associated with increased expression of Bcl2 and decreased apoptosis.