We have previously demonstrated that high glucose, glucosamine and growth factors increase extracellular matrix (ECM) protein synthesis, such as laminin and fibronectin, in renal mesangial cells and that the transcription factor cyclic AMP responsive element (CRE) binding protein CREB may participate in their expression. However, the molecular mechanism(s) of ECM gene regulation in mesangial cells still remains unknown. Laminin g1 and fibronectin genes are known to contain GC and CRE cis-elements in their promoters. In this study, we investigate the role of GC binding transcription factor Sp1 and CREB in ECM gene expression using laminin g1 promoter reporter (LAMC1-Luciferase) and CRE-SEAP (secretary alkaline phosphatase) plasmids, and their potential involvement in laminin g1 and fibronectin mRNA expression. In a double-transfection experiment using the LAMC1-Luciferase and pcDNA-Sp1 vectors, we observe that Sp1 expression increases luciferase activity 4.0-fold in 24 h when compared to pcDNA3.1 controls. On the other hand, siRNA silencers targeted against Sp1 significantly reduces LAMC1-luciferase activity (2.5-fold) against the control. Similarly, transient expression of the pcDNA-CREB vector in CRE-SEAP transfected mesangial cells induces the SEAP activity in the culture medium 1.5-fold when compared to pcDNA3.1 controls while transfection of a dominant negative K-CREB plasmid inhibits CRE-SEAP activity significantly (2-fold versus the control). In addition, we found that CREB overexpression increases laminin g1 and fibronectin mRNA levels 3.2- and 1.9- fold against respective controls as revealed by real-time quantitative PCR while the expression of K-CREB reduces the level of both mRNAs. Furthermore, treatment of mesangial cells for 48 h with high glucose (25 mM), glucosamine (1.5 mM) or with IGF-1 (100 ng/ml for 2 h) increases the expression of laminin g1 and fibronectin messages in mesangial cells significantly, and K-CREB completely impedes their effects. These results suggest that transcription factors Sp1 and CREB are involved in the regulation of ECM gene expression in renal mesangial cells and that they may participate in ECM accumulation and basement membrane thickening in the diabetic kidney. The materials and methods developed in this study may provide a platform for a potential gene therapy of diabetes and other renal diseases in the future.