BACKGROUND: Colon cancer is often driven by mutations of the adenomatous polyposis coli (APC) gene, an essential tumor suppressor gene of the Wnt β-catenin signaling pathway. APC and its cytoplasmic interactions have been well studied. However, various groups have also observed its presence in the nucleus. Identifying novel interactions of APC in the Wnt pathway will provide an opportunity to understand APC's nuclear role better and ultimately identify potential cancer treatment targets. METHODS: We used the all-vs-all sequencing (AVA-Seq) method to interrogate the interactome of protein fragments spanning most of the 60 Wnt β-catenin pathway proteins. Using protein fragments identified the interacting regions between the proteins with more resolution than a full-length protein approach. Pull-down assays were used to validate a subset of these interactions. RESULTS: 74 known and 703 novel Wnt β-catenin pathway protein-protein interactions were recovered in this study. There were 8 known and 31 novel APC protein-protein interactions. Novel interactions of APC and nuclear transcription factors TCF7, JUN, FOSL1, and SOX17 were particularly interesting and confirmed in validation assays. CONCLUSION: Based on our findings of novel interactions between APC and transcription factors and previous evidence of APC localizing to the nucleus, we suggest APC may compete and repress CTNNB1. This would occur through APC binding to the transcription factors (JUN, FOSL1, TCF7) to regulate the Wnt signaling pathway including through enhanced marking of CTNNB1 for degradation in the nucleus by APC binding with SOX17. Additional novel Wnt β-catenin pathway protein-protein interactions from this study could lead researchers to novel drug designs for cancer.