Engagement of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells (ECs) by anti-ICAM coated beads generates vesiculization via cell adhesion molecule (CAM)-mediated endocytosis, a clathrin-/caveolae-independent pathway involving Na+/H+ exchanger 1 (NHE1). ICAM-1 itself plays a key role in transendothelial migration (TEM) of leukocytes, particularly via the transcellular route. This involves endothelial endocytic vesicles that coalesce into transmigration pores, through which leukocytes transmigrate without disrupting EC junctions. The contribution of CAM-mediated endocytosis to the formation of docking sites and vesicular structures supporting TEM was explored in this study. Results show that the ICAM-1/NHE1-dependent CAM-mediated pathway associates with acid sphingomyelinase and ceramide. This supports plasmalemma deformability and cytoskeleton rearrangement, bridging these events to the formation of endothelial docking structures and vesicles involved in leukocyte transmigration.