Phenethyl isothiocyanate (PEITC), an isothiocyanate abundantly found in cruciferous vegetables have been shown to induce apoptosis through MAPK pathway in prostate and colon cancer cells. In the present study, we investigate the effect of PEITC on cell cycle regulation of HT-29 colon cancer cells. Using flow cytometry and Western blot analyses, we found that PEITC significantly induced G1 cell cycle arrest in HT-29 cells. We showed that the cell cycle arrest was not related to beta-catenin translocation into the nucleus. Interestingly, inhibition of p38 attenuated the cell cycle arrest, suggesting that cell cycle arrest by PEITC was caused by the activation of MAPK pathway. Treatments of PEITC resulted in a dose-dependent down-regulation of cyclins A, D, E and pRb protein expression. The down-regulation can be attributed to the activation of the p38 pathway, since inhibition of its activities by specific inhibitor blocked PEITC's ability to decrease the expression level of cyclins A and D and attenuate cell cycle arrest effect of PEITC. In conclusion, this study shows for the first time that PEITC can arrest HT-29 cells in G1 phase by down-regulation of cyclins through the activation of p38 MAPK signaling pathway.