Beta-catenin is implicated in quite different cellular processes, which require a fine-tuned regulation of its function. Here we demonstrate that cyclin-dependent kinase 6 (CDK6), in association with cyclin D1 (CCND1), directly binds to beta-catenin. We showed that CCND1-CDK6 phosphorylates beta-catenin on serine 45 (S45). This phosphorylation creates a priming site for glycogen synthase kinase 3beta (GSK3beta) and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Moreover, co-immunoprecipitation assays using Wnt3a-conditioned medium reveals that while Wnt stimulation leads to the dissociation of beta-catenin from axin and casein kinase Ialpha (CKIalpha), Wnt treatment promotes an increase in CCND1 level and the association of beta-catenin with CCND1-CDK6. Furthermore, Wnt3a-stimulated cytosolic beta-catenin levels were higher in CDK6 knockout mouse embryonic fibroblasts (CDK6-/- MEFs) compared to wild-type MEFs. Thus, the CCND1-CDK6 complex is like to negatively regulate Wnt signaling by mediating beta-catenin phosphorylation and its subsequent degradation in Wnt-stimulated cells.