Abstract Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.