Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.