We report that the cell surface mucin CD43 acts as an anti-adhesin on T lymphocytes. CD43-deficient murine lymphocytes homed significantly more frequently to secondary lymphoid organs than wild-type cells. Intravital microscopy of peripheral lymph node venules revealed that CD43-deficient lymphocytes were twice as likely to tether, roll, and stick than wild-type cells. This effect was due to CD43 interference with the homing receptor, L-selectin, and was most pronounced in venules with low L-selectin ligand density. In vitro, CD43-deficient cells tethered to L-selectin ligands more efficiently and rolled more slowly than wild-type lymphocytes. Thus, CD43 exerts a negative regulatory effect on T cell trafficking by counterbalancing L-selectin-mediated adhesion.