The expression of ephrinB2 is a prerequisite for the development of the embryonic vasculature and been established as a marker for the arterial endothelial cell phenotype. However, only little is known about the function of this ligand in mature arteries of the adult vasculature. In this context, our findings show that ephrinB2 is localized on the luminal endothelial surface and its expression is up-regulated during vascular remodelling processes, such as angiogenesis and arteriogenesis, but also atherosclerosis. Interestingly, the expression of the ephrinB receptor EphB2 on monocytes increases during differentiation into macrophages, and thereby potentially facilitating their interaction with ephrinB2-expressing endothelial cells during diapedesis. In fact, adhesion of monocytes to native arterial endothelial cells abundantly expressing ephrinB2 is strongly enhanced. Moreover, exposing monocytes to ephrinB2-coated surfaces induces forward signalling that leads to phosphorylation of EphB2 and an increased expression of pro-inflammatory chemokines such as IL-8 and MCP-1. On the other hand, reverse signalling is induced by stimulating endothelial cells with clustered EphB2/Fc which induces a JNK-dependent upregulation of the expression of adhesion molecules, such as VCAM-1 and E-selectin. Likewise, monocytes attachment to EphB2 pre-stimulated endothelial cells is reinforced. We conclude that the ephrinB/EphB system may affect the functional and structural integrity of the vessel wall during pro-inflammatory remodelling processes by simultaneously affecting monocyte and endothelial cell activity through forward and reverse signalling.