Caveolin-1 (Cav-1) is the principal component of plasma membrane caveolae that negatively regulates a number of cellular signaling events including canonical Wnt signaling. Activation of the Wnt/β-catenin pathway is essential for dorsal organizer formation and specification in early vertebrate embryos, but it remains not well understood what controls dorsal activity of maternal β-catenin and how Cav-1 functions in zebrafish embryogenesis. Here, we report that Cav-1 is required for proper dorsoventral patterning in zebrafish. Both Wnt and BMP signals act coordinately to negatively control transcriptional expression of cav-1 during embryonic development. Ectopic expression of Cav-1α or -1β resulted in formation of typical ventralized embryos, whereas Cav-1 knockdown led to abnormal embryos with expanded expression of dorsal genes. Cav-1 overexpression disrupts the nuclear translocation of β-catenin through the interaction of its scaffolding domain with Cav-1 binding motif of β-catenin. This reciprocal interaction is necessary for the ventralizing activity of Cav-1. We have further demonstrated that human Cav-1 proteins have conserved ventralizing activity in zebrafish embryogenesis. Thus, maternally expressed zebrafish Cav-1 regulates dorsoventral patterning by limiting nuclear translocation of active β-catenin. © 2010 .