Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-beta-TrCP (SCFbeta-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor beta (TGF-beta)-Smad3 pathway promotes SCF(beta-TrCP)-mediated Cdc25A ubiquitination. Cells treated with TGF-beta, as well as cells transfected with Smad3 or a constitutively active type I TGF-beta receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-beta-induced ubiquitination is associated with Cdc25A phosphorylation at the beta-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and beta-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-beta-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits beta-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-beta. beta-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFbeta-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-beta-Smad3 pathway.