TGF-β signalling can play a paradoxical cell type specific role in cancer progression. Smad4 is a key mediator of the TGF-β pathway, and is mutated and/or deleted in many cancers. To investigate Smad4 regulated TGF-β signalling in colon cancer we conducted an iTRAQ mass spectrometry quantitative screen using wild type SW480 (Smad4 negative) colon carcinoma cells and stably restored Smad4 positive SW480 cells. In cells possessing a restored canonical TGF-β signalling pathway, 48 h TGF-β stimulation induced the expression of 15 proteins and repressed 1 protein, while in Smad4 wild type cells, TGF-β induced 7 proteins and repressed 2 proteins. The expression of several S100 protein family members (A2, A4, A10, and A11), transgelin-2 and AKAP12, amongst others, were shown to be regulated by TGF-β in a Smad4 dependent manner. We observed that S100 A4 could be repressed by TGF-β, independently of Smad4 expression, while other Smad4 independent TGF-β responses were restricted to induction of ribosomes and cytoskeletal proteins. Our proteomic screen has identified new Smad4 dependent and independent TGF-β responses in colon carcinoma cells.