Lymphocyte homing is mediated by the interaction between L-selectin on lymphocytes and its glycoprotein ligands modified with 6-sulfo sialyl Lewis x (6-sulfo sLex) glycans on high endothelial venules (HEVs) in peripheral lymph nodes (PLNs). However, the lack of specific antibodies reactive with both human and mouse 6-sulfo sLex has limited our understanding of its function in vivo. Here, we generated a novel monoclonal antibody, termed SF1, that specifically reacts with 6-sulfo sLex expressed on HEVs in both species in a manner dependent on sulfate, fucose, and sialic acid modifications. Glycan array and biolayer interferometry analyses indicated that SF1 specifically bound to 6-sulfo sLex with a dissociation constant of 6.09 × 10–9 M. SF1 specifically bound to four glycoproteins from PLNs corresponding to the molecular sizes of L-selectin ligand glycoproteins. Consistently, SF1 inhibited L-selectin-dependent lymphocyte rolling on 6-sulfo sLex-expressing cells ex vivo and lymphocyte homing to PLNs and nasal-associated lymphoid tissues in vivo. Furthermore, SF1 significantly attenuated ovalbumin-induced allergic rhinitis in mice in association with significant suppression of Th2 immune responses. Collectively, these results suggest that SF1 can be useful for the functional analysis of 6-sulfo sLex and may potentially serve as a novel therapeutic agent against immune-related diseases.