Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.