In diabetes and hypertension, the induction of increased transforming growth factor-β (TGF-β) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-β activator thrombospondin-1 (TSP1). Because activation of latent TGF-β is a major means of regulating TGF-β, we addressed the role of TSP1-mediated TGF-β activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-β activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-β activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-β activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-β activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-β activation as a potential antifibrotic therapeutic strategy are warranted. Copyright © American Society for Investigative Pathology.