Axin antagonizes the developmental effects of Wnt in vertebrates. We show here that Axin simultaneously binds two components of the Wnt pathway, β-catenin and its negative regulator glycogen synthase kinase-3β. In mammalian cells, Axin inhibits Wnt-1 stimulation of β-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks β-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin, by forming a complex with β-catenin and glycogen synthase kinase-3β, can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations.