Vitamin K2 (VK2) has been shown to have a potent anti-tumor effect against several cancer types including hepatocellular carcinoma (HCC), but the mechanisms remain to be elucidated. Matrix metalloproteinase (MMP) plays an important role in the invasion and metastasis of cancer cells, but it is not known whether VK2 regulates the expression of MMPs. Human HCC cell lines were treated with VK2 combined with 12-O-tetradecanoyl phorbol-13 acetate (TPA) and the expression of MMPs was examined by reporter gene assay, RT-PCR and Western blotting. VK2 inhibited the basal and TPA-induced expression of MMP-1, -3 and -7 at the transcriptional, mRNA and protein levels in a dose-dependent manner. VK2 also inhibited the TPA-induced activation of NF-kappaB and AP-1 activity. The inhibitors against NF-kappaB and mitogen-activated protein kinases (MAP kinase) including ERK and JNK pathways suppressed TPA-induced luciferase activity of MMP-1, -3 and -7 promoters. These data suggest that VK2 inhibits MMP expression by suppressing NF-kappaB and MAP kinase activity and might be potentially useful in the treatment of HCC.