Both antisense morpholino oligonucleotide (MO)‐mediated knockdown and overexpression experiments were performed to analyze zebrafish cdx1b's function in intestinal cell differentiation. Substantial reductions in goblet cell numbers were detected in intestines of 102‐ and 120‐hours post‐fertilization (hpf) cdx1b MO‐injected embryos (morphants) compared to cdx1b‐4‐base mismatched (4mm)‐MO‐injected and wild type embryos. A significant decrease in enteroendocrine cell numbers was also observed in intestines of 96‐hpf cdx1b morphants. Furthermore, ectopic cdx1b expression caused notable increases in respective cell numbers of enteroendocrine and goblet cells in intestines of 96‐ and 98‐hpf injected embryos. Decreased PepT1 expression was detected in enterocytes of intestines in cdx1b morphants from 80 to 102 hr of development. In addition, increased cell proliferation was detected in intestines of cdx1b morphants. Overall, our results suggest that zebrafish cdx1b plays important roles in regulating intestinal cell proliferation and the differentiation of various intestinal cell lineages. Developmental Dynamics 238:1021–1032, 2009. © 2009 Wiley‐Liss, Inc.