In vertebrate development, Wnt/β-catenin signaling has an early role in specification of dorsal/anterior identity and a late one in posterior specification. To understand the evolution of these roles, we cloned β-catenin from the invertebrate chordate amphioxus. The exon/intron organization of β-catenin is highly conserved between amphioxus and other animals including a cnidarian, but not Drosophila. In development, amphioxus β-catenin is concentrated in all nuclei from the 16-cell stage until the onset of gastrulation when it becomes undetectable in presumptive mesendoderm. Li+, which up-regulates Wnt/β-catenin signaling, had no detectable effect on axial patterning when applied before the late blastula stage, suggesting that a role for β-catenin in specification of dorsal/anterior identity may be a vertebrate innovation. From the mid-gastrula through the neurula stage, the highest levels of nuclear β-catenin are around the blastopore. In the early neurula, β-catenin is down-regulated in the neural plate, but remains high in adjacent non-neural ectoderm. Embryos treated with Li+ at the late blastula stage are markedly posteriorized and lack a neural plate. These results suggest that in amphioxus, as in vertebrates, down-regulation of Wnt/β-catenin signaling in the neural plate is necessary for maintenance of the neuroectoderm and that a major evolutionarily conserved role of Wnt/β-catenin signaling is to specify posterior identity and pattern the anterior/posterior axis. Developmental Dynamics 233:1430–1443, 2005. © 2005 Wiley-Liss, Inc.