Siglecs are key immunoreceptors in immune homeostasis and cancer immunosuppression. Diverse endogenous and exogenous sialoglycan ligands induce Siglec-mediated immunoregulation, but the nature of endogenous Siglec ligands and how seemingly ubiquitous sialoglycans achieve selectivity is unclear. Here, we employed a HEK293 cell-based array and glycoprotein reporters designed from human mucins and mucin-like proteins with O-glycans in natural clusters and repeat motifs to dissect specificities of Siglec-7 and −15. We first used precomplexed Siglec-Fc chimera to demonstrate that CHST1-mediated 6- O -sulfation markedly enhanced binding to select O-glycoproteins, namely MAdCAM1, CD43, and PSGL-1. Probing binding properties of Siglec-7 expressed exogenously on CHO cells or endogenously on human monocytes by reverse assays employing fluorophore-tagged O-glycoprotein reporters revealed specific binding to the sulfo-sialyl core1 O-glycoform of the same O-glycoproteins. Our study indicates that Siglec-7 on immune cells employs multivalent interactions with repeated motifs of clustered sulfo-sialyl O-glycans to drive selectivity in ligand interactions and Siglec-induced immunosuppression.