AbstractHeparin is a highly sulfated glycosaminoglycan (GAG) of natural origin used as an anticoagulant and antithrombotic drug. These properties are principally based on the binding and activation of antithrombin (AT) through the pentasaccharide sequence GlcNAc/NS,6S‐GlcA‐GlcNS,3,6S‐IdoA2S‐GlcNS,6S (AGA*IA). Literature data show that the population of the ²S₀ ring conformation of the 2‐O‐sulfo‐α‐l‐iduronic acid (IdoA2S) motif correlates with the affinity and activation of AT. It was recently demonstrated that two synthetic AGA*IA‐containing hexasaccharides (one G unit added at the reducing end), differing in the degree of sulfation of the IdoA unit, show comparable affinity and ability to activate AT, despite a different conformation of the IdoA residue. In this paper, the binding of these two glycans to AT was studied by isothermal titration microcalorimetry (ITC), transferred (tr‐) NOESY, saturation transfer difference (STD) NMR spectroscopy and molecular dynamics (MD) simulations. Results indicated that both the IdoA2S and the IdoA units assume a ²S₀ conformation when bound with AT, and so present a common binding epitope for the two glycans, centred on the AGA*IA sequence.