Neuropilin-1 (NRP1) is a cell-surface receptor for both vascular endothelial growth factor(165) (VEGF(165)) and class 3 semaphorins that is expressed by neurons and endothelial cells. NRP1 is required for normal developmental angiogenesis in mice. The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries. The zebrafish NRP1 gene (znrp1) was isolated and when overexpressed in cells, zNRP1 protein was a functional receptor for human VEGF(165). Whole-mount in situ hybridization showed that transcripts for znrp1 during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Morpholino-mediated knockdown of zNRP1 in embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels. Circulation via trunk axial vessels was not affected. Embryos treated with VEGF receptor-2 kinase inhibitor had a similar intersegmental vessel defect suggesting that knockdown of zNRP1 reduces VEGF activity. To determine whether NRP1 and VEGF activities were interdependent in vivo, zNRP1 and VEGF morpholinos were coinjected into embryos at concentrations that individually did not significantly inhibit blood vessel development. The result was a potent inhibition of blood cell circulation via both intersegmental and axial vessels demonstrating that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results provide the first physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway.