Hepatocellular carcinoma (HCC) is a common human cancer with high mortality, and currently, there is no effective chemoprevention or systematic treatment. Recent evidence suggests that cyclooxygenase-2 (COX-2)-derived PGE(2) and Wnt/beta-catenin signaling pathways are implicated in hepatocarcinogenesis. Here, we report that omega-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) inhibit HCC growth through simultaneously inhibition of COX-2 and beta-catenin. DHA and EPA treatment resulted in a dose-dependent reduction of cell viability with cleavage of poly ADP ribose polymerase, caspase-3, and caspase-9 in three human HCC cell lines (Hep3B, Huh-7, HepG2). In contrast, AA, a omega-6 PUFA, exhibited no significant effect. DHA and EPA treatment caused dephosphorylation and thus activation of GSK-3beta, leading to beta-catenin degradation in Hep3B cells. The GSK-3beta inhibitor, LiCl, partially prevented DHA-induced beta-catenin protein degradation and apoptosis. Additionally, DHA induced the formation of beta-catenin/Axin/GSK-3beta binding complex, which serves as a parallel mechanism for beta-catenin degradation. Furthermore, DHA inhibited PGE(2) signaling through downregulation of COX-2 and upregulation of the COX-2 antagonist, 15-hydroxyprostaglandin dehydrogenase. Finally, the growth of HCC in vivo was significantly reduced when mouse HCCs (Hepa1-6) were inoculated into the Fat-1 transgenic mice, which express a Caenorhabditis elegans desaturase converting omega-6 to omega-3 PUFAs endogenously. These findings provide important preclinical evidence and molecular insight for utilization of omega-3 PUFAs for the chemoprevention and treatment of human HCC.