Stanniocalcin-1 (STC-1) is a glycoprotein hormone originally identified as a regulator of calcium and phosphate homeostasis in bony fish. Up-regulation of the mammalian homolog in numerous gene profiling studies of angiogenesis and vascular endothelial growth factor-A (VEGF-A(165))-regulated gene expression, suggests that regulation of this factor may be a key feature of the angiogenic response. Here we investigated the mechanisms mediating VEGF-A(165)-induced STC-1 gene expression in human endothelial cells. VEGF-A(165), acting via VEGFR2/KDR, induced STC-1 through de novo transcription, mediated primarily via intracellular protein kinase C (PKC)- and extracellular signal-regulated protein kinase (ERK)-dependent pathways. VEGF-A(165)-induced STC-1 mRNA expression was synergistically enhanced up to 2-fold by co-treatment with FGF-2, in a mechanism dependent on VEGFR2/KDR and FGFR1. Production of STC-1 protein by endothelial cells was also induced by VEGF-A(165) and synergistically enhanced by co-treatment with FGF-2. Synergism between VEGF-A(165) and FGF-2 was mediated via a novel neuropilin-1 (NP-1)-dependent mechanism, as indicated by the complete inhibition of synergism with either EG3287, a specific neuropilin antagonist, or siRNA-mediated NP-1 knockdown, and by the inability of the VEGF-A(121) isoform to synergise with FGF-2. Surprisingly, we found that NP-1 knockdown also markedly reduced KDR expression in HUVECs, and enhanced the VEGF-A(165)-induced reduction in KDR expression resulting from receptor-mediated endocytosis. These findings support a role for NP-1 in mediating synergistic effects between VEGF-A(165) and FGF-2, which may occur in part through a contribution of NP-1 to KDR stability.