Although casein kinase Iepsilon (CKIepsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIepsilon activates the Wnt signaling pathway in co-operation with Dvl. CKIepsilon and Axin associated with different sites of Dvl, and CKIepsilon and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIepsilon alone did not accumulate beta-catenin, but their co-expression accumulated greatly. Dvl and CKIepsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIepsilon activated Tcf, it did not synergize with CKIepsilon. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIepsilon. Furthermore, Dvl and CKIepsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dvl and CKIepsilon synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIepsilon to Axin is necessary for their synergistic action.