Lymphocyte homing is mediated by specific interaction between L-selectin on lymphocytes and the carbohydrate ligand 6-sulfo sialyl Lewis X on high endothelial venules. Here we generated mice lacking both core 1 extension and core 2 branching enzymes to assess the functions of O-glycan-borne L-selectin ligands in vivo. Mutant mice maintained robust lymphocyte homing, yet they lacked O-glycan L-selectin ligands. Biochemical analyses identified a class of N-glycans bearing the 6-sulfo sialyl Lewis X L-selectin ligand in high endothelial venules. These N-glycans supported the binding of L-selectin to high endothelial venules in vitro and contributed in vivo to O-glycan-independent lymphocyte homing in wild-type and mutant mice. Our results demonstrate the critical function of N-glycan-linked 6-sulfo sialyl Lewis X in L-selectin-dependent lymphocyte homing and recruitment.