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• PEITC inhibits the invasion and migration of colorectal cancer cells. • PEITC suppresses the EMT of colorectal cancer cells. • PEITC blocks the TGF-β1/Smad signaling pathway and TGF-β1 induced EMT. • PEITC might serve as a novel therapeutic agent with potent anti-cancer effects. Tumor metastasis is the leading cause of death in patients with colorectal cancer (CRC), in which epithelial-mesenchymal transition(EMT) plays a vital role. However, the exact mechanisms of this process remain largely unknown. The aim of the present study was to determine the role of phenethyl isothiocyanate (PEITC) in CRC metastasis by regulating EMT. Wound healing assays and Transwell matrix assays were used to evaluate the potential of PEITC to inhibit CRC cells invasion and migration in vitro. Western blotting, light microscopy and immunofluorescence assays were used to detect the occurrence of EMT. Luciferase activity assay, real time-PCR and western blotting were used to investigate TGF-β1/Smad signaling activity. We observed that PEITC, an isothiocyanate compound from crucifer with chemopreventive potential, inhibited the invasion and migration of CRC cells. Moreover, we showed that PEITC regulated the EMT of CRC cells. Additionally, we demonstrated that PEITC blocked the activation of the TGF-β1/Smad pathway and significantly suppressed TGF-β1-induced EMT. Our results suggested that PEITC plays a crucial role in inhibiting the invasion and migration of CRC cells by regulating TGF-β1-induced EMT. The results of the present study provide a theoretical basis for the use of PEITC to treat CRC. [ABSTRACT FROM AUTHOR]