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Allyl isothiocyanate (AITC) is a constituent of cruciferous vegetables that exhibits antitumor activity. In this study, AITC was shown to inhibit the proliferation of human metastatic colorectal adenocarcinoma SW620 cells in vitro by inducing cell cycle arrest at the G2/M phase. The signaling pathway of AITC action involved the down-regulation of the pivotal Cdc25B and Cdc25C protein phosphatases in the treated cells. Quantitative real-time PCR of AITC-treated SW620 cells revealed a time-dependent down-regulation of Cdc25B and Cdc25C mRNA levels, which resulted in a decrease in the expression levels of these two proteins. Upon prolonged exposure, AITC induced caspase-mediated apoptosis in SW620 cells. The apoptotic process was evidenced by the activation of initiator caspases (-8 and -9) and effector caspases (-3 and -7), and the cleavage of poly(ADP-ribose) polymerase (PARP). The antitumor activity of AITC was further demonstrated in a SW620 xenograft in vivo. Taken together, the results suggest that AITC is a potential candidate for future research in chemoprevention and chemotherapy.