D-bifunctional protein (DBP) is a highly conserved enzyme involved in peroxisomal beta-oxidation. We investigated the role of DBP during zebrafish embryogenesis. Zebrafish dbp is expressed early during development. Morpholino-mediated Dbp knockdown generates embryo with small head, pericardial edema and short tail. In agreement with its involvement in peroxisomal fatty acid oxidation, yolk lipid consumption is highly reduced in Dbp knockdown embryos. Endoderm-derived organs such as liver and pancreas are almost absent in morphants. Dbp knockdown also affects other aspects of animal development, such as neuronal maturation, cartilage formation, blood cell development and vasculogenesis. Consistent with morphological defects upon Dbp knockdown, significant changes in the expression of various genes involved in ether phospholipid synthesis, mitochondrial biogenesis, peroxisomal protein import and beta-oxidation pathway in peroxisome. Taken together, these results indicate that Dbp deficiency causes severe developmental abnormalities some of which has not been reported yet in human or mouse studies, and suggest that zebrafish can be used as an animal model to study organelle functions in vivo.