Excessive transforming growth factor-β (TGFβ) activity in hyperglycemia contributes to the development of diabetic nephropathy. Glucose stimulation of TGF-β activity and matrix synthesis are dependent on autocrine thrombospondin 1 (TSP1) to convert latent TGF-β to its biologically active form. The mechanisms by which glucose regulates TSP1 are not known. High glucose inhibits nitric oxide (NO) bioavailability and decreased NO increases TGF-β activity and extracellular matrix accumulation. Yet, the impact of NO signaling on TSP1 activation of TGF-β is unknown. We tested the role of NO signaling in the regulation of TSP1 expression and TSPI-dependent TGF-β activity in rat mesangial cells exposed to high glucose. On exposure to 30 mM glucose, NO accumulation in the conditioned media and intracellular cGMP levels were significantly decreased. The addition of an NO donor prevented the glucose-dependent increase in TSP1 mRNA, protein, and TGF-β bioactivity. The effects of the NO donor were blocked by ODQ (a soluble guanylate cyclase inhibitor) or Rp-8-pCPT-cGMPS (an inhibitor of cGMP-dependent protein kinase). These effects of high glucose were also reversed by the nitric-oxide synthase cofactor tetrahyrobiopterin (BH4). These results show that high glucose mediates increases in TSP1 expression and TSP1-dependent TGF-β bioactivity through down-modulation of NO-cGMP-dependent protein kinase signaling.