Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.