Phenethyl isothiocyanate (PEITC), one of the major compounds from dietary cruciferous vegetables, has been found to have antitumor properties and therefore could generate special interest for the development of chemopreventive and/or chemotherapeutic agent for human cancers. In the primary studies, we found that PEITC induced cytotoxic effect (decreased the percentage of viable cells) in human colon cancer HT29 cells. Here, in this study, we are the first to report the antimetastatic effect of PEITC in HT29 human colon cancer cells. The results show that PEITC exhibited an inhibitory effect on the abilities of adhesion, migration, and invasion by Boyden chamber assay. Western blotting examination indicated that PEITC exerted an inhibitory effect on the SOS-1, PKC, ERK1/2 and Rho A for causing the inhibitions of MMP-2 and -9 then followed by the inhibition of invasion and migration of HT29 cells in vitro. PEITC also affected Ras, FAK, PI3K or inhibited GRB2, NF-κB, iNOS and COX-2 for causing the inhibition of cell proliferation in HT29 cells. Real-time PCR also showed that PEITC inhibited the gene expressions of MMP-2, -7, -9, FAK and Rho A after PEITC treatment for 48 h in HT29 cells. PEITC also inhibited the activities of AKT, ERK, JNK and PKC. Our results provide a new insight into the mechanisms and functions of PEITC which inhibit migration and invasion of HT29 human colon cancer cells. These results suggest that molecular targeting of NF-κB led to the inhibition of MMP-2, -7, and -9 and it might be a useful strategy for the inhibition of migration and invasion on human colon cancer.