Duplin binds to -catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the -catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin ␣ was isolated as a binding protein of Duplin. Importin ␣ bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin ⌬500 -584 ) retained Duplin in the cytoplasm. Duplin ⌬500 -584 bound to -catenin as efficiently as wild-type Duplin, but it neither repressed Wntdependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a -catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed -catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin ␣, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream -catenin target genes.