BACKGROUND Autologous vein grafting remains the gold standard for surgical bypass grafts. However, vein bypasses still have significant incidence of failure. Ephrin type-B receptor 4 (EphB4), the embryonic venous determinant, may modulate vein graft adaptation. Although EphB4 is expressed in venous endothelial and smooth muscle cells (SMCs), it is not known whether EphB4 is functional in human SMCs. METHODS Human adult venous SMCs were obtained from the inferior vena cava of an adult human liver donor. Primary SMCs were stimulated with EphrinB2/Fc or transfected with an EphB4-expression vector (GV219-EphB4). Expression of SMC phenotype markers, migration, and proliferation were evaluated. RESULTS Activation of EphB4 with EphrinB2/Fc increased the mRNA and protein expression of the venous SMC contractile markers alpha actin, calponin-1, SM22, and MYH11, while decreasing the expression of the synthetic marker osteopontin. EphrinB2/Fc treatment inhibited SMC migration, but not proliferation. In addition, overexpression of EphB4 increased mRNA expression of SMC contractile markers, while decreasing expression of the apoptosis marker caspase-9. CONCLUSIONS EphB4 was present and functional in adult human venous SMCs. Stimulation of EphB4 increased expression of contractile SMC phenotypic markers and decreased SMC migration in vitro, functioning to retain the contractile phenotype of SMCs. EphB4 activation, therefore, recapitulates changes observed during vein graft adaptation to the arterial environment in vivo. EphB4 represents a new strategy to inhibit neointimal hyperplasia during vein graft adaption.