The Wnt pathway controls numerous developmental processes via the ␤-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of ␤-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic ␤-catenin associates with APC and axin and is continuously phosphorylated by GSK-3␤, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3␤ from phosphorylating ␤-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of ␤-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces ␤-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate ␤-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3␤ and is both necessary and sufficient to initiate the ␤-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.