UNLABELLED: AXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.