The loss of growth-inhibitory responses to transforming growth factor-β (TGF-β) is a frequent consequence of malignant transformation. Smad2, Smad3, and Smad4 proteins are important mediators of the antiproliferative responses to TGF-β and may become inactivated in some human cancers. Epithelial cells harboring oncogenic Ras mutations often exhibit a loss of TGF-β anti-proliferative responses. To further investigate the effect of oncogenic Ras in TGF-β signaling, we used an isopropyl-1-thio-β-D-galactopyranoside-inducible expression system to express Ha-RasVal-12 in intestinal epithelial cells. Induction of Ha-RasVal-12 caused a decrease in the level of Smad4 expression, inhibited TGF-β-induced complex formation between Smad2/Smad3 and Smad4, blocked Smad4 nuclear translocation, inhibited the TGF-β-mediated decrease in [3H]thymidine incorporation, and repressed TGF-β-activated transcriptional responses. The withdrawal of isopropyl-1-thio-β -D-galactopyranoside or the addition of an inhibitor of the ubiquitin-proteasome pathway restored the Smad4 level and TGF-β-induced Smad complex formation. Forced expression of Smad4 resulted in partial recovery of the TGF-β-mediated growth inhibition and transcriptional responses in the presence of oncogenic Ras. Further, PD98059, a specific inhibitor of the MEK/ERK/mitogen-activated protein kinase pathway prevented the Ras-induced decrease in Smad4 expression and complex formation. Our results suggest a novel mechanism by which oncogenic Ras represses TGF-β signaling by mitogen-activated protein kinase-dependent down-regulation of Smad4, thereby subverting the tumor suppressor function of TGF-β.