Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in β1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-β (TGF-β) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-β-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-β-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-β signaling pathway.