The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class VI Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class VI Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent non-tumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assay both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.