Nuclear factor kappa B (NF-κB) has been implicated in cancer cell survival. We explored the role of the TGF-β pathway in the regulation of NF-κB in colon cancer cells. TGF-β-1 treatment of the colon adenocarcinoma cell line FET-1, results in an early increase in IκB-α phosphorylation that precedes NF-κB nuclear translocation and DNA binding activity. Activation of the TGF-β type I receptor is required for the TGF-β-mediated activation of NF-κB. No activation of NF-κB is observed in a Smad4 null cell line, SW480, even though TGF-β does result in IκB-α phosphorylation in these cells. Smad4 restores the TGF-β-1-mediated NF-κB activation in SW480 cells. TGF-β-1 treatment fails to activate NF-κB or phosphorylate IκB-α in FET-1 cells expressing the inhibitory Smad, Smad7. Taken together, these results suggest a role for Smad4 in the transcriptional activation of NF-κB, and a direct effect of Smad 7 inhibiting IκB-α phosphorylation rather than through the well-established inhibition of Smad2/3 phosphorylation with subsequent inhibition of the TGF-β pathway. © 2005 Elsevier Inc. All rights reserved.