2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a major component of Polygonum multiflorum Thunb (He-Shou-Wu), has been reported to exhibit antioxidant and anti-inflammatory effects. However, its anti-metastatic effect against colorectal cancer is still unclear. In this study, cell migration, invasion and adhesion abilities as well as metastasis-associated protein and NF-κB pathway signaling factor expression were analyzed after treating HT-29 cells with THSG. According to the results, the migration and invasiveness of HT-29 cells were reduced after treatment with 5 or 10 mM THSG (p<0.05). Additionally, the levels of matrix metalloproteinase-2 (MMP-2) and phosphorylated VE-cadherin in HT-29 cells were reduced and the transepithelial electrical resistance (TEER) of EA.hy926 endothelial cell monolayers was increased after incubation in THSG for 24 h (p<0.05). Cell adhesion ability and the E-selectin and intercellular adhesion molecule-1 (ICAM-1) protein levels were reduced when EA.hy926 endothelial cells were treated with THSG (p<0.05). In addition, the cytoplasmic phosphorylation of IκB, the nuclear p65 level and the DNA-binding activity of NF-κB were reduced after treating HT-29 or EA.hy926 cells with 5 or 10 mM THSG (p<0.05). These results suggest that THSG inhibits HT-29 cell metastasis by suppressing cell migration, invasion and adhesion. Furthermore, THSG inhibits metastasis-associated protein expression by suppressing NF-κB pathway activation.