Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent commonly found in edible cruciferous vegetables. It has been implicated also for therapy, and is in clinical trial for lung cancer. Here, we provide evidence that the tumor suppressive effect of PEITC is related to its ability to induce expression of damaged DNA binding protein 2 (DDB2), a DNA repair protein involved also in apoptosis and premature senescence. DDB2 expression is attenuated in a wide variety of cancers including the aggressive colon cancers. We show that, in colon cancer cells, reactive oxygen species, which are induced by PEITC, augment expression of DDB2 through the p38MAPK/JNK pathway, independently of p53. PEITC-induced expression of DDB2 is critical for inhibition of tumor progression by PEITC. Tumors derived from DDB2-deficient colon cancer cells are refractory to PEITC-treatments, resulting from deficiencies in apoptosis and senescence. The DDB2-proficient tumors, on the other hand, respond effectively to PEITC. The results show that PEITC can be used to induce expression of DDB2, and that expression of DDB2 is critical for effective response of tumors to PEITC.