Aberrant WNT signal transduction is involved in many diseases. In colorectal cancer and melanoma, mutational disruption of proteins involved in the degradation of β-catenin, the key effector of the WNT signaling pathway, results in stabilization of β-catenin and, in turn, activation of transcription. We have used tandem-affinity protein purification and mass spectrometry to define the protein interaction network of the β-catenin destruction complex. This assay revealed that WTX, a protein encoded by a gene mutated in Wilms tumors, forms a complex with β-catenin, AXIN1, β-TrCP2 (β-transducin repeat-containing protein 2), and APC (adenomatous polyposis coli). Functional analyses in cultured cells, Xenopus, and zebrafish demonstrate that WTX promotes β-catenin ubiquitination and degradation, which antagonize WNT/β-catenin signaling. These data provide a possible mechanistic explanation for the tumor suppressor activity of WTX.