It has been shown that Ring finger protein 43 (RNF43) is overexpressed in colorectal cancer and mediates cancer cell proliferation; however, its role in hepatocellular carcinoma (HCC) remains unknown. In this study, we found that RNF43 was frequently overexpressed in HCCs, and this overexpression was correlated with positive vascular invasion, poor tumor differentiation, and advanced tumor stage. Functional studies showed that knockdown of RNF43 could induce apoptosis and inhibit proliferation, invasion, colony formation, and xenograft growth of HCCs. Microarray-based gene profiling showed a total of 229 genes differentially expressed after RNF43 knockdown, many of which are involved in oncogenic processes such as cell proliferation, cell adhesion, cell motility, cell death, DNA repair, and so on. These results suggest that RNF43 is involved in tumorigenesis and progression of HCCs and that antagonism of RNF43 may be beneficial for HCC treatment.