The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left–right (L–R) axis in mouse and zebrafish. However, the role of Brachyury in L–R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L–R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L–R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant-negative type Xbra (Xbra-EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal-related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra-GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L–R specification may be the direct regulation of Zic3 expression.