Galectin-3 (Gal-3) is a multifunctional effector acting extracellularly after non-classical secretion, in the cytoplasm and the nucleus. Its modular display of a carbohydrate recognition domain (CRD) attached to a tail of collagen-like repeats (nine in the human protein) and an N-terminal peptide is unique in this lectin family and not yet fully explored, as is the physiological significance of serine and tyrosine phosphorylation. Using a series of nine synthetic (phospho)peptides and the 15N-labeled CRD of human Gal-3 as well as measuring chemical shift perturbations in mixtures, potential for peptide reactivity was revealed in Gal-3’s backface. Tyrosine phosphorylation reduced the affinity, while serine phosphorylation of the N-terminal peptide was essential. Controls with sequence scrambling underscored inherent specificity. These results detect capacity for distinct sites of intramolecular recognition in Gal-3, adjustable by phosphorylation, and thus prompt analysis using the full-length protein. ; 11 p. ; We gratefully acknowledge the financial support by MINECO of Spain (Grant CTQ2012-32025) and Comunidad de Madrid (MHit project) as well as the EC-funded BM1003 and CM1102 COST actions,the GlycoHIT program (contract No. 260600) and the GLYCOPHARM ITN project. M.A.B. acknowledges a FPI Ph.D. fellowship from the Spanish Ministry of Economy and Competitiveness.