Neuropilins (NRPs) are 130 kDa receptors that bind and respond to the class-3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell-binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 nM and 3.9 nM respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A E291A) in the 280-290’s b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E N290D and R287E N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect the SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.