Abstract Background Sorafenib is currently the only approved systemic agent for advanced hepatocellular carcinoma (HCC); however, its efficacy is limited. Aberrant activation of WNT/β-catenin signaling pathway has been shown to mediate resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway would improve the anti-tumor effect of sorafenib in HCC. Materials and Methods Human HCC cell lines, including Huh7, HepG2, PLC5, and Hep3B, were included. Inhibition of WNT/β-catenin pathway was achieved by ICG-001, a small molecule disrupting the interaction of cAMP-responsive element binding (CREB)-binding protein (CBP) and β-catenin and inhibiting the β-catenin-mediated transcription activities, or by RNA interference (RNAi)- downregulation of β-catenin. The efficacy of sorafenib combined with WNT/β-catenin pathway inhibition in HCC cells was determined by MTT for cell viability, and by flow cytometry and Western blotting for apoptosis. The in vivo efficacy was evaluated in a subcutaneous xenograft mode of Huh7 cells. Results In multiple HCC cell lines, ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently; the combination of ICG-001 and sorafenib showed a synergistic antitumor effect. Downregulation of β-catenin by RNAi increased sorafenib sensitivity, whereas overexpression of β-catenin decreased sorafenib sensitivity in Huh7 cells. Furthermore, the sorafenib-sensitization effect by short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was offset by β-catenin overexpression. Mechanistically, sorafenib in combination of ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a more prominent downregulation of Mcl-1 in HCC cells. Finally, ICG-001 in combination of sorafenib showed a more significantly growth-retarding effect in Huh7 xenograft than either drug alone. Conclusion Our data indicate that inhibition of WNT/β- catenin signaling pathway improves the antitumor effect of sorafenib against HCC in vitro and in vivo.(This study was supported by grants NRPB-100CAP1020-2 and NSC-101-2314-B-002-141, 100CAP1020-2). Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2015-5336