The VEGF family comprises seven members that are designated VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PlGF), and VEGF-F. Of these factors, VEGF-D plays important roles for angiogenesis and lymphangiogenesis, and could promote tumor growth and lymphatic metastasis. In this study, we identified a zebrafish VEGF-D homolog that encodes a 272 amino acid protein including a PDGF (platelet-derived growth factor) domain characteristic to VEGF family. Expression profile demonstrated that the VEGF-D began expressed from 13 somite stage. Microinjecting zVEGF-D mRNA into zebrafish 1-cell stage embryos resulted in severe misguidance of intersegmental vessels (ISV) and abnormal connection between dorsal aorta and caudal vein. Microangiography indicated that these abnormal ISVs were not functional. Our studies therefore identified the first non-mammalian VEGF-D and established its in vivo role for vascular system development during vertebrate embryogenesis and provided an alternative animal model to further reveal functions of VEGF-D.