Abstract: Using triple quadrupole mass spectrometry, the gas-phase protonated cytosine could be generated by elctrospraying a water-methanol (50∶50, V/V) solution of cytidine with the higher declustering potential. Collision-induced dissociation (CID) of protonated cytosine has four competing dissociation channels, corresponding to the eliminate NH₃ (-17 u) to form the ion m/z 95, loss of HNCO (-43 u) and H₂O (-18 u) to form the ions m/z 69 and m/z 94, and loss of C₂NH₃ (-41 u) to form the ion m/z 71. These primary product ions were further confirmed by their relative fragmentations through CID. Ab initio and density function theory (DFT) calculation were also employed to simulate the whole process. About 9 stable protonated cytosine isomers were optimized and the potential energy surfaces (PESs) for the isomerization processes were explored in detail. With enough energy, isomerization can occur firstly from one structure to the other, and then based on the various isomer structures, the extra energy will drive the dissociation to happen. So the different initial structure will lead to the different dissociation channel to produce different product ions. The four primary and further dissociation channels of protonated cytosine were also theoretically investigated, and the energy barriers for the primary dissociation to eliminate the molecule NH₃, HNCO, H₂O and C₂NH₃ are 292.2, 355.0, 586.6 and 838.6 kJ/mol, respectively, which are consistent with the peak abundant of the energy-resolved CID of protonated cytosine to form the ions m/z 95, 69, 94, 71.