The highly anionic synthetic pentasaccharide fondaparinux (FDPX) - representing the antithrombin binding sequence of heparin - is in clinical use as a potent anticoagulant. Contrary to the unfractionated heparin, FDPX lacks potent antidote completely reversing its anticoagulant activity, therefore it is of great importance to identify new structures exhibiting strong intermolecular interactions towards FDPX. The polycationic heptakis(6-amino-6-deoxy)-beta-cyclodextrin (NH2-β-CD) can serve as an excellent model compound to mimic these interactions between the oppositely charged oligosaccharides. Herein, extensive NMR spectroscopic and nano-electrospray ionization mass spectrometric (nESI-MS) studies were conducted to understand the molecular-level interactions in the FDPX - NH2-β-CD systems. NMR experiments were performed at pD 7.4 and 2.0. Job's method of continuous variation and 1H NMR titration experiments suggested the formation of FDPX∙NH2-β-CD complex at pD 7.4, while the presence of multiple complexes was assumed at pD 2.0. Stability constants were determined by separate 1H NMR titrations, yielding log β11=3.65 ± 0.02 at pD 7.4, while log β11 ≥ 4.9 value suggested a high-affinity system at pD 2.0. 2D NOESY NMR studies indicated spatial proximities between the anomeric resonance α-l-iduronic acid residue and the cyclodextrin's methylene unit in the proximity of the cationic amino function. Acidic degradation of FDPX was investigated by NMR and MS for the first time in detail confirming that desulfation occurs involving one to two sulfate moieties. The desulfation of FDPX was inhibited by the cationic cyclodextrin in the case of equimolar ratio at pD 2.0. This is the first report on the stabilizing effect of cyclodextrin complexation on heparin degradation.