Triptolide/PG490 (Trp), an extract of the Chinese herb Tripterygium wilfordii Hook f, is a potent anti-inflammatory agent that has entered Phase I clinical trials for solid tumors. While its anti-proliferative effects are well-characterized, the possible anti-migratory effects of Trp have not been described. We have recently demonstrated that curcumin and emodin, two other anti-inflammatory herbal extracts, significantly decrease colon cancer cell migration in vitro; emodin was recently shown to do so in a phosphatidylinositol-3-kinase (PI3K) dependent manner. The purpose of this study was to: (i) determine if Trp inhibits colon cancer cell migration, and (ii) examine the possible mechanisms of its anti-migratory action. METHODS. (i) Migration of HCT116 cells on collagen was assessed using a transwell chamber system. Cells were treated with low doses of Trp, emodin, and LY294002 (a specific PI3K inhibitor). EGF or neurotensin (NT) were used as chemoattractants. (ii) Western blotting was used to detect changes in phosphorylated Akt (pAkt) protein expression. The effect of Trp on NFAT and NF-κB DNA-binding activity was analyzed by gel shift assays. Ribonuclease protection assays were used to examine mRNA expression of VEGF, c-myc, and TGF-β family receptors. RESULTS. (i) Trp reduces basal and stimulated HCT116 migration by 65-80%, compared with 25-40% by emodin and 40-60% by LY294002. (ii) Trp treatment decreased pAkt expression at 1 and 4 h, which correlates with reported effects of PI3K/Akt on colon cancer cell migration. Trp treatment of HCT116 cells decreased DNA binding of NFAT, but not NF-κB, and potently inhibited expression of VEGF, c-myc, and TGF-β Receptor type I. CONCLUSIONS. Trp is a potent inhibitor of colon cancer cell migration in vitro. This effect may be partially mediated through the PI3K pathway, a mechanism which has not been previously described. Importantly, we demonstrate for the first time, TGF-βR downregulation by Trp; this effect, in combination with inhibition of NFAT binding, may contribute to the anti-metastatic action of this natural herbal extract.