AbstractWe investigated two recently synthesized and characterized sialyl derivatives, bearing the Neu5Ac‐α‐(2‐6)‐Gal epitope, as promising binders for Siglec‐7, an inhibitory Siglec mainly found on natural killer cells. A variety of sialoglycan structures can be recognized by Siglec‐7 with implications in the modulation of immune responses. Notably, overexpression of sialylated glycans recognized by Siglec‐7 can be associated with the progression of several tumors, including melanoma and renal cell carcinoma. NOE‐based NMR techniques, including Saturation Transfer Difference and transferred‐NOESY NMR, together with molecular docking and dynamic simulations were combined to shed light on the molecular basis of Siglec‐7 recognition of two conformationally constrained Sialyl‐Tn antigen analogs. We, therefore, identify the ligands epitope mapping and their conformational features and propose 3D models accurately describing the protein‐ligand complexes. We found that the binding site of Siglec‐7 can accommodate both synthetic analogs, with the sialic acid mainly involved in the interaction. Moreover, the flexibility of Siglec‐7 loops allows a preferred accommodation of the more rigid compound bearing a biphenyl moiety at position 9 of the sialic acid that contributed to the interaction to a large extent. Our findings provided insights for developing potential novel high affinity ligands for Siglec‐7 to hinder tumor evasion.