The expression of ephrinB2 in endothelial cells is a prerequisite for adequate remodelling processes of the developing or angiogenic vasculature. We studied the role of this arterial marker gene in remodelling processes of adult arterial blood vessels by analyzing arterioles undergoing arteriogenic remodelling. These experiments revealed a significant up-regulation of ephrinB2 in endothelial cells. To investigate the functional role of ephrinB2 during vascular remodelling, we conducted experiments using endothelial cell-smooth muscle cell co-culture spheroids. By analyzing the spheroidal organization we found that ephrinB2 enhances the segregation of both cell types. Furthermore, ephrinB2 inhibits smooth muscle cell migration and may thus restrict migration of these cells within defined borders during the remodelling processes of the vessel wall. Interestingly, detailed confocal immunohistochemical analyses suggest that ephrinB2 is expressed on the luminal endothelial cell surface. Subsequently, we analyzed the effects of ephrinB2 on leukocyte transmigration through an endothelial cell monolayer which was inhibited by over-expression of non functional ephrinB2 in the endothelial cells. Taken together, these results suggest that up-regulation of ephrinB2 during arterial remodelling processes affects migration of smooth muscle cells as well as leukocyte diapedesis and may thus context-dependently affect the plasticity of the vessel wall.