Cellulosic materials present as tissue, paper, wood, or filter materials in low and intermediate level waste will degrade under alkaline conditions if water ingresses in a cementitious backfilled repository. The main degradation product is isosaccharinic acid. Complex formation with isosaccharinic acid may adversely affect the retention of radionuclides by the sorption or formation of solid phases. Hence, this compound is of particular concern in the context of nuclear waste disposal. Structural information of complexes is limited to spherical metal centers and little is known about the interaction of uranyl (UVIO22+) with isosaccharinic acid. Therefore, the interaction of UO22+ with α-isosaccharinate (ISA) was studied under acidic conditions focusing particularly on the structural characterization of the formed complexes. Attenuated total reflection Fourier-transform infrared (ATR-FTIR), nuclear magnetic resonance (NMR), UV-Vis, extended X-ray absorption fine structure (EXAFS) spectroscopy and electrospray-ionization mass spectrometry (ESI-MS) were combined with theoretical calculations to obtain a process understanding on the molecular level. The dominant binding motifs in the formed complexes are 5- and 6-membered rings involving the carboxylic group as well as the α- or β-hydroxy group of ISA. Two concentration dependent complex formation mechanisms were identified involving either mono- ([UO2(ISA)(H2O)3]+) or binuclear ([(UO2)2(ISA)(H2O)6]3+) species. Furthermore, this study unveils the interaction of UO22+ with the protonated α-isosaccharinic acid (HISA) promoting its transformation to the corresponding α-isosaccharinate-1,4-lactone (ISL) and inhibiting the formation of polynuclear UO22+–ISA species. Future studies on related systems will benefit from the comprehensive knowledge concerning the behavior of ISA as a complexing agent gained in the present study.