Wnt signaling mediated by β-catenin plays crucial roles in the development of hepatocellular carcinoma and other cancers such as colorectal cancer. β-Catenin associates with T-cell factor (TCF) transcription factors and functions as a transcriptional activator in the nucleus. By protein interaction screening, we identified EBP50, a cytoplasmic protein with 2 PDZ domains, as a β-catenin-associating molecule. EBP50 interacted with β-catenin through its carboxyl-PDZ domain in vitro and in vivo. Northern blot and RT-PCR analysis revealed an increase of EBP50 messenger RNA (mRNA) in hepatocellular carcinoma (HCC) cell lines and surgical specimens of human HCC. Over-expression of EBP50 protein with focal nuclear localization was detected in human HCC. In human HCC and colorectal cancer cell lines, EBP50 enhanced β-catenin/TCF-dependent transcription in a dose-dependent manner. In an HCC cell line, over-expression of the carboxyl PDZ domain resulted in a decrease of endogenous β-catenin/TCF transactivation. EBP50 promoted β-catenin-mediated transactivation only in cells in which β-catenin was already stabilized, suggesting that EBP50 may work with stabilized β-catenin for transcriptional regulation. In conclusion, the EBP50/β-catenin complex promotes Wnt signaling, and over-expression of EBP50 may work cooperatively with β-catenin in the development of liver cancer.