AbstractThe human macrophage galactose‐type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N‐acetylgalactosamine (GalNAc) containing structures present on pathogens, self‐glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc‐containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL‐CRD, C181‐H316) in the absence and presence of methyl α‐GalNAc (α‐MeGalNAc), a simple monosaccharide, shows that the MGL‐CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL‐CRD structure explains the ability of MGL to accommodate different GalNAc‐containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2‐derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.