Nepmucin/CLM-9 is an Ig domain-containing sialomucin that is expressed in vascular endothelial cells. Our studies demonstrate that, similar to CD31, nepmucin is localized at interendothelial junctions and vesicle-like structures along the cell border, where it undergoes intracellular recycling. Functional analyses reveal that nepmucin mediates both homotypic and heterotypic cell adhesion through its Ig domain. Endothelial cells expressing nepmucin exhibit enhanced lymphocyte transendothelial migration (TEM), which can be inhibited by anti-nepmucin monoclonal antibodies (mAbs) that block either homophilic or heterophilic binding. Notably, mAbs that inhibit homophilic binding are able to block TEM without affecting lymphocyte adhesion. These findings suggest that endothelial nepmucin facilitates lymphocyte TEM through multiple adhesion pathways.