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)-β-related ligands that regulate cell growth and differentiation. Mutations in Smad4/ DPC4 have been identified in ∼50% of pancreatic adenocarcinomas. Here we report that SCFβ-TrCP1, a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein β-TrCP1 in this E3 ligase interacted with Smad4 and that SCFβ-TrCP1 inhibited TGF-β 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 β-TrCP1 and significantly elevated protein ubiquitination by SCFβ-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 β-TrCp1. Both Smad4 levels and TGF-β signaling were elevated by retrovirus-delivered β-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.