We have reported previously that acyclic retinoid, a synthetic retinoid X receptor α (RXRα)-ligand, suppresses the development of hepatocellular carcinoma (HCC) in patients with chronic liver disease. On the other hand, HCCs become refractory to physiological concentrations of the natural RXRα-ligand, 9-cis retinoic acid (9cRA), due to extracellular signal-regulated kinase (Erk) 1/2-mediated phosphorylation and inactivation of RXRα. Here, we show that acyclic retinoid restores the function of RXRα in human HCC-derived HuH7 cells by inactivating the Ras-Erk 1/2 signaling system, thereby dephosphorylating RXRα. In contrast, 9cRA failed to suppress phosphoErk 1/2 levels and subsequent RXRα phosphorylation. Although 9cRA also suppressed Ras activity, it simultaneously down-regulated mitogen-activated protein kinase phosphatase-1, an enzyme that inactivates Erk, thereby leaving the phosphorylation status of Erk unchanged. A combination of 9cRA (a potent ligand) and acyclic retinoid (a weak ligand preventing phosphorylation) resulted in a marked cooperation in transactivation via the RXR-response element and in inhibiting the proliferation of HuH7 cells. These events provide a novel molecular basis for the antitumor activity of acyclic retinoid against HCC.