The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (DeltaC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated DeltaC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to DeltaC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptor-overexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor-ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, full-length ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium.