The activation of oncogenes and the inactivation of tumor suppressor genes by mutations or chronic hepatitis virus infections play key roles in the pathogenesis of hepatocellular carcinoma (HCC). Here we report that RN181, a really interesting new gene finger domain-containing protein, was down-regulated in highly malignant cell lines and in tumor cells of 139 HCC clinical samples in comparison with adjacent normal liver tissues. The expression of RN181 was strongly associated with the pathological grade of HCC. Alterations of the expression of RN181 by retrovirus-transduced up-regulation and short hairpin RNA-mediated down-regulation demonstrated the function of RN181 as a tumor suppressor because it decreased the proliferation and colony formation of HCC cells in vitro and inhibited tumor growth in vivo by suppressing cell proliferation and enhancing cell apoptosis in xenografted tumors. Proteomic analyses showed that RN181 regulates the expression of many proteins that are important in many cellular processes. Statistical analyses identified 33 proteins with consistent changes (≥2-fold) in RN181-transformed cells. Ten of these proteins were up-regulated by RN181, and 23 were down-regulated. Representative proteins were validated by western blotting. Interaction network investigations revealed that 20 RN181-regulated proteins could integrate several key biological processes such as survival, metabolism, and mitogen-activated protein kinase (MAPK) pathways. Remarkably, 11 of the 33 proteins are associated with MAPK signaling in one or more ways. RN181 suppressed the tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in cell lines and in tumor cells of xenografts and HCC clinical samples, and removing the suppression increased tumor growth. CONCLUSIONS We have shown that RN181 suppresses the tumorigenesis of HCC through the inhibition of ERK/MAPK signaling in the liver. Our results provide new insights into the pathogenesis of HCC and may help with the development of novel therapeutic strategies.