The C-type lectin receptor Langerin is a glycan binding protein that serves as an uptake receptor on Langerhans cells and is essential for the formation of Birbeck granules. Whereas most of Langerin´s ligands are recognized via a canonical Ca2+-dependent binding site, interactions with heparins have been proposed to make additional contacts to a secondary, Ca2+-independent site. Glycan array screening and biomolecular NMR spectroscopy were employed to investigate the molecular mechanism of these interactions. We observed that binding of heparin hexasaccharides to a secondary site does not require the presence of Ca2+ and activates a previously identified intradomain allosteric network of Langerin