The c-myc protooncogene is a key regulator of cell proliferation whose expression is reduced in normal epithelial cells in response to the growth inhibitory cytokine TGF-β. Smad4 mediates this inhibitory effect of TGF-β by forming a complex with Smad3, E2F4/5, and p107 at the TGF-β inhibitory element (TIE) element on the c-myc promoter. In contrast, cell proliferation and c-myc expression are increased in response to Wnt ligands; this effect is mediated through the lymphoid enhancer-binding factor 1/T cell-specific factor (LEF/TCF) family of transcription factors on the c-myc promoter LEF/TCF-binding elements (TBE1 and TBE2). We report that a peptide aptamer designed to inhibit the binding between Smad4 and LEF/TCF reduced c-myc expression and the growth rate of HepG2 cells. Further analysis demonstrated that, in the absence of TGF-β, Smad4 was bound to the positive regulatory element TBE1 from the c-myc promoter and activated c-myc promoter activity. Smad4 binding to the positive TBE1 c-myc element was reduced by TGF-β, consistent with Smad4's inhibitory role on c-myc expression in response to TGF-β. Reduction of Smad4 levels by RNAi knockdown also reduced c-myc expression levels and sensitized hepatocytes to cell death by serum deprivation. Two tumor-derived mutant Smad4 proteins that fail to mediate TGF-β responses were still competent to cooperate with LEF1 to activate the c-myc promoter. These results support a previously unreported TGF-β-independent function for Smad4 in cooperating with LEF/TCF to activate c-myc expression.