Uncontrolled activation of transforming growth factor (TGF)-β results in a wide range of pathologic conditions. Therapeutic interventions to regulate TGF-β signaling during fibrosis have been developed but the effectiveness is still limited. Here, we show that developmental endothelial locus-1 (Del-1) ameliorates fibrosis in mice by inhibiting α_v integrin-mediated activation of TGF-β. Del-1 bound to α_vβ₆ integrin, an important activator of TGF-β, and inhibited the binding of α_vβ₆ integrin to the latency-associated peptide (LAP), thereby suppressing α_v integrin-mediated activation of TGF-β. Lack of Del-1 increased colocalization of α_v integrin and LAP in the lungs, which was reversed by Del-1 supplementation. The crucial role of Del-1 in regulating TGF-β activity was recapitulated in a mouse model of fibrosis using an adenovirus expressing inactive TGF-β1. Del-1 supplementation improved the pathological characteristics of the mice and reduced mortality. Thus, we propose that Del-1 is a negative regulator of TGF-β activation and a potential anti-fibrotic factor.