Expression of the arterial marker molecule ephrinB2 in endothelial cells is a prerequisite for adequate remodeling processes of the developing or angiogenic vasculature. Although its role in these processes has been extensively studied, the impact of ephrinB2 on the remodeling of adult arteries is largely unknown. To this end, we analyzed its expression during a biomechanically induced arteriolar remodeling process known as arteriogenesis and noted a significant increase in ephrinB2 expression under these conditions. By examining those biomechanical forces presumed to drive arteriogenesis, we identified cyclic stretch as a critical inducer of ephrinB2 expression in endothelial cells. Subsequent functional analyses in vitro revealed that endothelial cells expressing ephrinB2 limit the migration of smooth muscle cells, thereby enhancing segregation of both cell types. Moreover, MCP-1 induced transmigration of monocytes through a monolayer of endothelial cells overexpressing a truncated variant of ephrinB2 was clearly impeded. Taken together, these data suggest that expression of ephrinB2 in adult endothelial cells is up-regulated during arterial remodeling and controlled by cyclic stretch, a well-known inducer of such processes. This stretch-induced ephrinB2 expression may be pivotal for arteriogenesis as it limits smooth muscle cell migration within defined borders and controls monocyte extravasation.