The pathophysiological mechanism involved in side effects of radiation therapy, and especially the role of the endothelium remains unclear. Previous results showed that plasminogen activator inhibitor-type 1 (PAI-1) contributes to radiation-induced intestinal injury and suggested that this role could be driven by an endothelium-dependent mechanism. We investigated whether endothelial-specific PAI-1 deletion could affect radiation-induced intestinal injury. We created a mouse model with a specific deletion of PAI-1 in the endothelium (PAI-1KO(endo)) by a Cre-LoxP system. In a model of radiation enteropathy, survival and intestinal radiation injury were followed as well as intestinal gene transcriptional profile and inflammatory cells intestinal infiltration. Irradiated PAI-1KO(endo) mice exhibited increased survival, reduced acute enteritis severity and attenuated late fibrosis compared with irradiated PAI-1(flx/flx) mice. Double E-cadherin/TUNEL labeling confirmed a reduced epithelial cell apoptosis in irradiated PAI-1KO(endo). High-throughput gene expression combined with bioinformatic analyses revealed a putative involvement of macrophages. We observed a decrease in CD68(+)cells in irradiated intestinal tissues from PAI-1KO(endo) mice as well as modifications associated with M1/M2 polarization. This work shows that PAI-1 plays a role in radiation-induced intestinal injury by an endothelium-dependent mechanism and demonstrates in vivo that the endothelium is directly involved in the progression of radiation-induced enteritis.