Digestive organ development occurs through a sequence of morphologically distinct stages in vertebrate; however, the underlying molecular mechanisms are largely unknown, especially in the expansion growth of the entire digestive system at the later stage. Here we present zebrafishdefhi429 mutant with a loss-of-function mutation in def (digestive-organ expansion factor). def is a novel pan-endoderm-specific gene, and lack of def confers hypoplastic digestive organs. Characterization of the defhi429 mutant showed that the def mutation affected cell proliferation, not cell differentiation, in the developing digestive organs except the endocrine pancreas at the later stage of endoderm organogenesis. Two strategies were applied to study the underlying molecular mechanism. Downstream gene expression profiling revealed that the selectively up-regulated expression of Δ113p53, a newly identified isoform of p53, is enriched within the mutant digestive organs, and induces the expression of p53-responsive genes to trigger the arrest of the cell cycle but not apoptosis, resulting in compromised organ growth in the mutant. The def gene encodes a nuclear-localized protein, a yeast two-hybrid screen was performed to identify Def interacting proteins which may function in the regulation of the growth of the digestive organs, and 16 positive candidates were obtained. Whole-mount in situ hybridization showed that 15 candidates are enriched in digestive organs during embryogenesis. To gain further insight into the biological functions of these Def interacting proteins, we designed gene-specific morpholinos to individually knock-down five selected genes appbp2, L159, L221, L245 and rybp in developing zebrafish embryos. While knock-down of appbp2, L159, L221 and L245 cause a phenotype mimicking the phenotype of defective digestive organs in the defhi429 mutant; the rybp morpholino injection does not cause obvious phenotype in the wild type embryos but could partially rescue ifabp expression in the intestine in the defhi429 mutant. Co-IP showed that Def and Rybp physically interact with each other in vivo. These results suggest that Appbp2, L159, L221 and L245 might form a complex with Def, either individually or collectively, to control the endoderm organogenesis whilst Rybp is probably a repressor for the development of digestive organs.