Isosaccharinic acid, a major final product of cellulose degradation under highly alkaline cement porewater conditions, is known to increase the mobility of actinides via strong complex formation. In this study, the formation of Am(III) complexes with α-d-isosaccharinate (ISA) was studied in terms of thermodynamics and coordination structures by combining spectrophotometry, time-resolved laser fluorescence spectroscopy (TRLFS), and density functional theory (DFT) calculations. The formation constants of the Am(III)-ISA complexes were determined by absorption spectroscopy at temperatures in the range of 15-70 °C. The measured reaction enthalpy and entropy changes indicate that the formation of a 1:1 Am(III)-ISA complex is driven by an increase in entropy. By contrast, the 1:2 complex formation is exothermic with a much less increase in entropy. DFT calculations predict that C2- and C4-hydroxyl groups, along with the carboxyl group, participate in the tridentate chelate binding of the primary ISA. The thermodynamic, TRLFS, and DFT results collectively suggest the tridentate binding of the primary ISA to Am(III) via a carboxylate and C2- and C4-hydroxyl groups in the protonated state and reduced dentate binding of the secondary ISA, such as bidentate binding, forming a four-membered ring structure via the carboxylate group.