BACKGROUND & AIMS: The beta-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of beta-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. METHODS: Plasmids encoding tagged full-length beta-catenin (CTNNB1) or beta-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reversetranscription polymerase chain reaction. Tagged full-length and mutant forms of beta-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of beta-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. RESULTS: Mice injected with plasmids encoding K335I or N387K beta-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT beta-catenin and MET. K335I and N387K beta-catenin bound APC with lower affinity than WT beta-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by beta-catenin. Studies of protein structures supported the observed changes in relative binding affinities. CONCLUSION: Expression of beta-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in beta-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of beta-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase beta-catenin signaling in cancer cells.