Cucurbitacin B (CuB) is an active component isolated from various plants used as folk medicine in Asian countries and has shown diverse antitumor activities. There is, however, no documented effect of CuB on the migration and invasion of human hepatoma cells yet. The purpose of this study was to assess the effect of CuB on the migration and invasion of hepatoma cells and to explore the possible mechanism.   Human hepatoma cell lines HepG2 and BEL-7402 were used for the study. Effects of CuB on cancer cell migration and invasion were evaluated in vitro with wound healing and transwell assays. The effect of CuB on the expression of matrix metalloproteinase (MMP)-9, mitogen-activated protein kinases (MAPKs), Akt, nuclear factor-κB (NF-κB), c-Fos and c-Jun was investigated with gelatin zymography and/or western blotting.   Cucurbitacin B has significantly suppressed 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell invasion and migration in a concentration-dependent manner, which was accompanied with suppression of TPA-induced MMP-9 expression through inactivation of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 and Akt. In the nucleus, it has also strongly suppressed TPA-stimulated expression of NF-κB, c-Jun and c-Fos.   Cucurbitacin B has a potential value for suppressing metastasis of human hepatoma cells through suppressing the expression of MMP-9. © 2011 The Japan Society of Hepatology.