Smad proteins are crucial for the intracellular signaling of transforming growth factor-beta (TGF-beta). After receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate transcription of a select set of target genes. Here, we investigated the turnover of Smad3, positively regulating Smad for TGF-beta signaling. In a steady state, the inhibition of proteasome activity leads to stabilization of Smad3 protein. Smad proteins are multi-ubiquitinated and degraded independently of the phosphorylation induced by the TGF-beta receptors. Moreover, the degradation of Smad3 was enhanced by treatment with TGF-beta, and phosphorylated Smad3 was accumulated on proteasome inhibition. Ubiquitination of phosphorylated Smad3 but not Smad3(3SA), a receptor-mediated phosphorylation-incompetent mutant, was observed in the nucleus after treatment with TGF-beta. These findings suggest that, in a steady state, Smad3 is constitutively degraded via the ubiquitin-proteasome pathway in the cytoplasm and that, in response to TGF-beta, it is phosphorylated and translocated into the nucleus, where it is degraded through the ubiquitin-proteasome pathway.