Metastable and prompt fragmentation of the deprotonated monosaccharide d-fructose was studied. By using the isotope labelled molecules 1-13C-d-fructose, 2-13C-d-fructose and 6-13C-d-fructose the origin of the observed fragments could unambiguously be identified. It was found that prompt fragmentation (<200 ns) is characterized by pronounced site selectivity, i.e. the neutral fragments contain C6 and the negative charge remains on the fragment containing the anomeric centre (C2). The selectivity is preserved in metastable dissociation (≈8 μs) only for the generation of the fragment ion C4H4O3− (m/z 100). For all other metastable decay channels fragments with charge retention on the C6 containing fragment contribute appreciably. Density functional theory (DFT) and classical dynamics simulations are applied to predict the formation of fragment ions. Most of the experimentally detected fragment ions were also observed in the simulations thereby revealing detailed information about the fragmentation pathways. We found that the deprotonation site strongly influences the pathway of the dissociation reactions.