Smad7 functions as an intracellular antagonist in transforming growth factor-beta (TGF-beta) signaling. In addition to interacting stably with the activated TGF-beta type I receptor (TbetaRI) to prevent phosphorylation of the receptor-regulated Smads (Smad2 and Smad3), Smad7 also induces degradation of the activated TbetaRI through association with different E3 ubiquitin ligases. Using the two-hybrid screen, we identified atrophin 1-interacting protein 4 (AIP4) as an E3 ubiquitin ligase that specifically targets Smad7 for ubiquitin-dependent degradation without affecting the turnover of the activated TbetaRI. Surprisingly, we found that despite the ability to degrade Smad7, AIP4 can inhibit TGF-beta signaling, presumably by enhancing the association of Smad7 with the activated TbetaRI. Consistent with this notion, expression of a catalytic mutant of AIP4, which is unable to induce ubiquitination and degradation of Smad7, also stabilizes the TbetaRI.Smad7 complex, resulting in inhibition of TGF-beta signaling. The ability of AIP4 to enhance the inhibitory function of Smad7 independent of its ubiquitin ligase activity reveals a new mechanism by which E3 ubiquitin ligases may function to turn off TGF-beta signaling.