Abstract c-myc promoter silencing is a key step in epithelial cell growth inhibition by transforming growth factor β (TGFβ). During carcinogenesis, however, epithelial cells escape from c-myc repression and consequently become refractory to TGFβ-mediated antiproliferation. Here, we assessed the role of the repressor, KLF11, in TGFβ-induced growth inhibition in normal epithelial as well as pancreatic carcinoma cells. Endogenous KLF11 was stably down-regulated by RNA interference technology, and the functional consequences were studied by proliferation assays, reporter assays, DNA binding studies, and expression analyses. Coimmunoprecipitation and glutathione S-transferase pulldown assays were conducted to define KLF11-Smad3 interaction and U0126 was administered to examine the effects of the extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase on complex formation and c-myc promoter binding of KLF11 and Smad3 in pancreatic cancer cells. In TGFβ-stimulated normal epithelial cells, nuclear KLF11, in concert with Smad3, binds to and represses transcription from the core region of the TGFβ-inhibitory element (TIE) of the c-myc promoter. Disruption of KLF11-Smad3 interaction or small interfering RNA–mediated knockdown of endogenous KLF11 strongly diminishes Smad3-TIE promoter binding and repression, and consequently impairs TGFβ-mediated growth inhibition. In pancreatic cancer cells with oncogenic Ras mutations, hyperactive ERK counteracts TGFβ-induced c-myc repression and growth inhibition through at least two mechanisms, i.e., via disruption of KLF11-Smad3 complex formation and through inhibition of KLF11-Smad3 binding to the TIE element. Together, these results suggest a central role for KLF11 in TGFβ-induced c-myc repression and antiproliferation and identifies a novel mechanism through which ERK signaling antagonizes the tumor suppressor activities of TGFβ in pancreatic cancer cells with oncogenic Ras mutations. (Mol Cancer Res 2006;4(11):861–72)