TGF-beta regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-beta has been reported to mediate stimulatory action of TGF-beta-induced ECM. Although TNF-alpha was reported to suppress theTGF-beta-induced CTGF gene expression, the molecular mechanism is not well clarified. In this study, we found the inhibitory effect of TNF-alpha on TGF-beta-induced CTGF expression in WT but not p65-/-MEF cells. TNF-alpha neither induced Smad7 expression nor affected TGF-beta-induced Smad2 phosphorylation and nuclear translocation. We demonstrated that p300 physically associated with p65 rather than Smad4 in the presence of both TNF-alpha and TGF-beta. Moreover, the TGF-beta-induced binding of p300 and acetylated H4, but not Smad4 to the CTGF promoter was disturbed by TNF-alpha treatment. Overall, our data showed that suppression of TNF-alpha on TGF-beta-induced CTGF expression is due to the competition of p300 by p65 and Smad4.