Thrombospondin-1 (TSP-1) inhibits angiogenesis and activates latent TGF-β1, both of which are strongly associated with progression of renal disease. Recently, it was reported that Smad2 but not Smad3 regulates TSP-1 expression in response to TGF-β1 in rat tubular epithelial cells as well as in mouse fibroblasts. This study investigated the role of ERK1/2 and p38 mitogen-activated protein kinases (MAPK). TGF-β1 activated both ERK1/2 and p38 in the rat proximal tubular cell line NRK52E. Blocking ERK1/2 and p38 inhibited TGF-β1-induced TSP-1 mRNA and protein expression. Next, the cross-talk between Smad2 and ERK1/2 or p38 was examined. Whereas blocking of ERK1/2 or p38 failed to inhibit TGF-β1-induced Smad2 activation, inhibition of Smad2 by Smad7 overexpression inhibited the phosphorylation of ERK1/2 but not p38 in response to TGF-β1. Similar results were observed using mouse fibroblasts from Smad2 knockout embryos, in that TGF-β1 was able to activate p38 but not ERK1/2 in this cell line. In conclusion, TSP-1 expression is regulated by both ERK1/2 and p38 MAPK in rat proximal tubular cells and mouse fibroblasts in response to TGF-β1. The ERK1/2 activation is dependent on Smad2 activation, whereas the p38 activation occurs independent of Smad2. Because TSP-1 is a major antiangiogenic molecule and an activator of TGF-β1, this provides an important insight to the mechanism by which TGF-β1 may mediate interstitial fibrosis and progressive renal disease.