Significance Siglecs are immunomodulatory receptors that recognize sialic acid–carrying glycans with important functions in immunity. However, many of their natural ligands are poorly defined. We generated a cell-based glycan array comprised of a library of isogenic human cells displaying the greater complexity of sialic acids on diverse glycan structures and glycoconjugates in the natural context of the cell surface. We applied this array to reveal fine binding specificities of Siglecs for sialoglycans, informed of the underlying required glycosyltransferase genes, and provided evidence for selective binding context provided by glycan presentation on distinct protein sequences. Insight into the fine binding specificities of Siglecs will advance understanding their diverse biological functions and benefit therapeutic targeting in autoimmunity, inflammation, cancer, and Alzheimer’s disease.