Axin uses different combinations of functional domains in down-regulation of the Wnt pathway and activation of the MEKK1/JNK pathway. We are interested in the elucidation of the functional switch of Axin. In the present study, we show that the Wnt activator CKIε, but not CKIIα, Frat1, LRP5, or LRP6, inhibited Axin-mediated JNK activation. We also found that both CKIα and CKIε interacted with Axin, whereas CKIIα did not bind to Axin and had no effect on Axin-mediated JNK activity even though CKIIα has also been suggested to be an activator for the Wnt pathway. The COOH-terminal region and the MEKK1-interacting domain of Axin are important for CKIα-Axin and CKIε-Axin interaction. We further demonstrated that CKIε and CKIα binding to Axin excluded MEKK1 binding, indicating that a competitive physical occupancy may underlie the inhibitory effect. Moreover, our data indicated that CKIε kinase activity plays an additive role in this effect. Taken together, we have demonstrated that CKI and CKII exhibit differential effects on Axin-MEKK1 interaction and Axin-mediated JNK activation. Furthermore, our data suggest that CKI may provide a possible switch mechanism for Axin function in the regulation of Wnt and JNK pathways.