N-(2-Hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA, denoted as H3L in this work, and the three dissociable protons represent those of the three carboxylic groups) is a strong chelating ligand and plays an important role in the treatment and disposal of nuclear wastes as well as separation sciences of f-elements. In this work, the complexation of HEDTA with U(VI) was studied thermodynamically and structurally in aqueous solutions. Potentiometry and microcalorimetry were used to measure the complexation constants (298-343 K) and enthalpies (298 K), respectively, at I = 1.0 mol.L-I NaClO4. Thermodynamic studies identified three 1:1 U(VI)/HEDTA complexes with different degrees of deprotonation, namely, UO2(HL)(aq), UO2L-1, and UO2(H-1L)(2-), where H-1 represents the deprotonation of the hydroxyl group. The results indicated that all three complexation reactions are endothermic and driven by entropy Complexation of U(VI) with HEDTA only. Coordination modes of the three complexes were investigated by NMR and extended X-ray absorption fine structure spectroscopies. In the UO2(HL)(aq) complex, HEDTA holds a tridentate mode, and the coordination occurs to the end of the ethylenediamine backbone. Two oxygens of the two carboxylic groups and one nitrogen of the amine group participate in the coordination. In both UO2L- and UO2(H-1L)(2-), HEDTA holds a tetradentate mode and coordinates to U(VI) along the side of the ethylenediamine backbone. The difference is that in the UO2(H-1L)(2-) complex, the alkoxide form of the HEDTA hydroxyl group directly binds to the U(VI) atom, forming a highly strong chelation.