Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K2 (VK2) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9-cis retinoic acid (9cRA) plus VK2 in the HuH7 human HCC cell line. We found that the combination of 1.0 μM ACR or 1.0 μM 9cRA plus 10 μM VK2 synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK2 also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK2 alone inhibited phosphorylation of the retinoid X receptor (RXR)α protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal-regulated kinase phosphorylation. Moreover, the inhibition of RXRα phosphorylation by VK2 was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK2 markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK2 cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRα and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.