The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax down-regulated numerous nuclear factor-kappaB (NF-kappaB) target genes and decreased the binding of NF-kappaB to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-kappaB activity in vascular endothelial cells. Because NF-kappaB has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-kappaB-dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype.