To investigate the underlying mechanisms that transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) in tumor cells contributes to 5-Fu resistance. A series of experiments involving cell-viability and caspase-activity analyses, siRNA transfection, RNA isolation and quantitative-PCR (qPCR) assay, cell migration analysis, western blotting analysis of total protein and membrane protein were performed in this study. Mouse xenograft model was used to determine the effect of the PAR2 inhibitor in vivo. In this study, we found that protease-activated receptor-2 (PAR2) induction in 5-Fu therapy is correlated with TGF-β-mediated EMT and apoptosis resistance. PAR2 and TGF-β were both activated in response to 5-Fu treatment in vivo and in vitro, and whereas TGF-β inhibition sensitized CRC cells to 5-Fu and suppressed cell migration, PAR2 activation eliminated the effect TGF-β inhibition. Conversely, siRNA-mediated PAR2 depletion or PAR2 inhibition with a specific inhibitor produced a similar phenotype as TGF-β signal inhibition: 5-Fu sensitization and cell-migration suppression. Moreover, the results of xenograft experiments indicated that the PAR2 inhibitor can enhance cell killing by 5-Fu in vivo and suppress EMT signaling. Our results reveal the TGF-β effects require the coordinating action of PAR2, suggesting that PAR2 inhibition could a new therapeutic strategy to combat 5-Fu resistance in CRC.