Reduced EphB4 expression is observed during vein graft adaptation and is associated with increased venous wall thickening. These findings suggest that EphB4 may mediate normal adult venous endothelial cell (EC) function and vein graft adaptation. We therefore tested the functional significance of EphB4 using EC with genetically reduced EphB4 signaling. EC were isolated from EphB4(+/+) and EphB4(+/-) mice. In vitro function was assessed through EC proliferation, migration, nitric oxide (NO) synthesis, and chemokine production. A mouse vein graft model was used to correlate in vitro findings with in vivo vein grafts. Smooth muscle cells (SMC) were subjected to proliferation and migration assays using EphB4(+/+) and EphB4(+/-) EC-conditioned medium. EphB4(+/-) EC exhibited diminished proliferation (P < 0.0001, n = 6), migration (P < 0.0001, n = 3), and NO production (P = 0.0012, n = 3). EphB4(+/-) EC had increased VEGF-A mRNA (P = 0.0006, n = 6) and protein (P = 0.0106, n = 3) as well as increased secretion of VEGF-A (P = 0.0010, n = 5), PDGF-BB (P < 0.0001, n = 6), and TGF-β1 (P < 0.0001, n = 6). EphB4(+/-)-conditioned medium promoted SMC proliferation (P < 0.0001, n = 7) and migration (P = 0.0358, n = 3). Vein grafts and EphB4(+/-) EC showed similarity with regard to VEGF-A and eNOS mRNA and protein expression. In conclusion, reduced venous EC EphB4 function is associated with a proangiogenic and mitogenic phenotype. EphB4(+/-) EC have increased secretion of SMC mitogens and reduced NO production that correlate with the thickened neointima formed during vein graft adaptation. These findings suggest that EphB4 remains active in adult venous EC and that loss of EphB4 plays a role in vein graft adaptation.