Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6′-sulfo-sLe^X (Neu5Acα2–3[6-SO₄] Galβ1–4[Fucα1–3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6′-sulfo-sLe^X. Secondary analysis showed that related structures, sialyl-Lewis X (sLe^X) and 6-sulfo-sLe^X containing 6-GlcNAc-SO₄ showed much lower binding avidity, indicating significant contribution of 6-Gal-SO₄ on Siglec-F binding to 6′-sulfo-sLe^x. The lectin activity of Siglec-F on mouse eosinophils was “masked” by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6′‐sulfo-sLe^X structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6′-sulfo-sLe^X. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6′-sulfo-sLe^X ligand in eosinophil biology.