Advanced glycation end-products (AGEs) play a critical role in diabetic nephropathy by stimulating extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) is a potent inducer of ECM synthesis and increases in the diabetic kidneys. To determine the critical role of CTGF in AGE-induced ECM accumulation leading to diabetic nephropathy, rats were given AGEs by intravenous injection for 6 weeks. AGE treatment induced a significant renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) accumulation in glomeruli, and a mild renal dysfunction, as shown by increases in urinary volume and protein content. AGE treatment also caused significant increases in renal CTGF and transforming growth factor (TGF)-beta 1 mRNA and protein expression. Direct exposure of rat mesangial cells to AGEs in vitro significantly induced increases in fibronectin and Col IV production, which could be completely prevented by pretreatment with anti-CTGF antibody. AGE treatment also significantly increased both TGF-beta 1 and CTGF mRNA expression; however, inhibition of TGF-beta 1 mRNA expression by shRNA or neutralization of TGF-beta 1 protein by anti-TGF-beta 1 antibody did not significantly prevent AGE-increased expression of CTGF mRNA and protein. These results suggest that AGE-induced CTGF expression, predominantly through a TGF-beta 1-independent pathway, plays a critical role in renal ECM accumulation leading to diabetic nephropathy.