The stability against collisional dissociation of [uracil–Ca]2+ complexes has been investigated by combining nanoelectrospray ionization/mass spectrometry techniques and B3LYP/6-311++G(3df,2p)//B3LYP/6-31+G(d,p) density functional theory (DFT) calculations. The reactivity upon collision seems to be dominated by Coulomb explosion processes, since the most intense peaks in the MS/MS spectra correspond to singly-charged species (CaOH+ and [C4,H3,N2,O]+). Nevertheless, additional peaks corresponding to the loss of neutral species, namely [H,N,C,O] and H2O have been also detected. A systematic study of the CID spectra obtained with different labeled species, namely, 2-13C-uracil, 3-15N-uracil and 2-13C-1,3-15N2-uracil, concludes unambiguously that the loss of [H,N,C,O] involves exclusively atoms C2 and N3. Suitable mechanisms for these fragmentation processes are proposed through a theoretical survey of the corresponding potential energy surface. A comparison between these results and those reported for two other metal dications, namely Cu2+ and Pb2+, as well as for protonated uracil and uracil–M+ (M = Li, Na, K) complexes denotes the existence of significant differences and interesting similarities, among the various systems.