Overexpression of a lefty orthologue, Ci-lefty, caused a failure of neural tube closure in the protochordate ascidian Ciona intestinalis. The body bent dorsally, and anterior-posterior elongation was inhibited. A similar phenotype was observed in embryos treated with SB431542, an inhibitor of Nodal receptors, suggesting that Ci-Lefty antagonized Nodal signaling as reported in other deuterostome species. Overexpression of Ci-nodal also resulted in a similar phenotype, suggesting that a correct quantity and/or a spatial restriction of Nodal signaling are important for the neural tube to form. In addition to known Ci-Nodal target genes, orthologues of Zic (Ci-ZicL) and cdx (Ci-cdx) were activated by Ci-Nodal. Expression of a dominant negative Ci-cdx caused defects in neural tube formation similar to those obtained on treatment with SB431542 or overexpression of Ci-lefty. A regulatory cascade composed of Ci-Nodal, Ci-ZicL, and Ci-Cdx may play an important role in neural tube formation in the Ciona embryo.