The tetraprotonated form of the dioxatetraazamacrocycle, 6,19-dioxa-3,9,16,22-tetraaza[22.2.2.2
11,14]triaconta-1(26),11,13,24,27,29-hexaene, (H
4L
1)
4+, was used as the receptor for binding studies with carboxylate anionic substrates of different shapes, sizes, and charges [succinate (suc
2−), cyclohexanetricarboxylate (cta
3−), phthalate (ph
2−), isophthalate (iph
2−), terephthalate (tph
2−), and benezenetricarboxylate (btc
3−)]. Association constants were determined by potentiometry in aqueous solution at 298.2 K and 0.10 M KCl and by
1H NMR titration in D
2O. The strongest association was found for the btc
3− anion at 5–7 pH region. From both techniques it was possible to establish the binding preference trend of the receptor for the different substrates, and the
1H NMR spectroscopy gave important suggestions about the type of interactions between partners and the location of the substrates in the supramolecular entities formed. The effective binding constants at pH 6 follow the order: btc
3−>iph
2−>cta
3−≈ph
2−>tph
2−>suc
2−. All the studies suggest that the anionic substrates bind to the receptor via N–H
O
C hydrogen bonds and electrostatic interactions, and the aromatic substrates can also establish π–π stacking interactions. The crystal structures of (H
4L
1)
4+ and its supramolecular assemblies with ph
2− and tph
2− were determined by X-ray diffraction. The last two structures showed that the association process in solid state occurs via multiple N–H
O
C hydrogen bonds with the anionic substrate located outside the macrocyclic cavity of the receptor. Molecular dynamics simulations carried out for the association of (H
4L
1)
4+ with tph
2− and btc
3− in water solution established at atomic level the existence of all interactions suggested by the experimental studies, which act cooperatively in the binding process. Furthermore, the binding free energies were estimated and the values are in agreement with the experimental ones, indicating that the binding of these two anionic substrates occurs into the receptor cavity. However, the tph
2− has also propensity to leave the macrocyclic cavity and its molecular recognition can also happen at the top of the receptor.