Galectins are glycan-binding proteins translating the sugar-encoded information of cellular glycoconjugates into physiological activities, including immunity, cell migration, and signaling. Galectins also interact with non-glycosylated partners in the extracellular milieu, among which the pre-B cell receptor (pre-BCR) during B cell development. How these interactions might interplay with the glycan-decoding function of galectins is unknown. Here, we perform NMR experiments on native membranes to monitor Gal-1 binding to physiological cell surface ligands. We show that pre-BCR interaction changes Gal-1 binding to glycosylated pre-B cell surface receptors. At the molecular and cellular levels, we identify a 2,3-sialylated motifs as key targeted epitopes. This targeting occurs through a selectivity switch increasing Gal-1 contacts with a 2,3-sialylated polyN-acetyllactosamine upon pre-BCR interaction. Importantly, we observe that this switch is involved in the regulation of pre-BCR activation. Altogether, this study demonstrates that interactions to non-glycosylated proteins regulate the glycan-decoding functions of galectins at the cell surface.