Abstract Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-β (TGF-β) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-β-induced growth inhibition, there seems to be a Smad4-independent TGF-β signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-β-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-β stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-β. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-β signal pathway that did not involve Smad4.