BACKGROUND & AIMS: The ParaHox transcription factor Cdx2 is an essential determinant of intestinal phenotype in mammals throughout development, influencing gut function, homeostasis, and epithelial barrier integrity. Cdx2 expression demarcates the zones of intestinal stem cell proliferation in the adult gut, with deregulated expression implicated in intestinal metaplasia and cancer. However, in vivo analysis of these prospective roles has been limited because inactivation of Cdx2 in mice leads to preimplantation embryonic lethality. We used the zebrafish, a valuable model for studying gut development, to generate a system to further understanding of the role of Cdx2 in normal intestinal function and in disease states. METHODS: We isolated and characterized the zebrafish cdx1b ortholog and analyzed its function by antisense morpholino gene knockdown. RESULTS: We showed that zebrafish Cdx1b replaces the role of Cdx2 in gut development. Evolutionary studies have indicated that the zebrafish Cdx2 loci were lost following the genome-wide duplication event that occurred in teleosts. Zebrafish Cdx1b is expressed exclusively in the developing intestine during late embryogenesis and regulates intestinal cell proliferation and terminal differentiation. CONCLUSIONS: This work established an in vivo system to explore further the activity of Cdx2 in the gut and its impact on processes such as inflammation and cancer.