Endothelial cell-selective adhesion molecule (ESAM) is a member of the immunoglobulin superfamily and mediates homophilic adhesion between endothelial cells. ESAM has been shown to bind to membrane-associated guanylate kinase (MAGUK) with inverted domain structure 1 (MAGI-1), but the interaction between these molecules remains unknown. We investigated the role of ESAM in the subcellular localization of MAGI-1 and cell adhesion by means of transfection experiments using Chinese hamster ovary (CHO) cells. Overexpression of ESAM recruited MAGI-1 to the cell-cell contact area. The intracellular domain of ESAM was necessary for the recruitment of MAGI-1 to the cell contact area, but did not participate in the initial cell-cell adhesion. Cell dissociation assays revealed that colocalization of ESAM and MAGI-1 promoted actin polymerization through the postsynaptic density 95/discs large/zonula occludens-1 (PDZ) domain and resulted in firm cell-cell adhesion, which was inhibited by an actin polymerization inhibitor. When the cells attach to each other, colocalization of ESAM and MAGI-1 can lead to the actin polymerization at intracellular contacts. Interaction of ESAM with MAGI-1 activated RhoA, and ESAM-mediated MAGI-1 recruitment to the cell membrane and mature cell adhesion were inhibited by a RhoA inhibitor. These findings suggest that ESAM may regulate MAGI-1 recruitment to the cell contacts, and subsequently promote actin polymerization and mature cell-cell adhesion through a RhoA-dependent mechanism.