The mechanism for the effects of protonation and CS methylation on the electrophilic addition reaction of Cyt has been explored by means of CBS-QB3 and CBS-QB3/PCM methods. In the gas phase, three paths, two protonated paths (N3 and 02 protonated paths B and C) as well as one neutral path (path A), were mainly discussed, and the calculated results indicate that the reaction of the HSO3- group with neutral Cyt is unlikely because of its high activation free energy, whereas O2-protonated path (path C) is the most likely to occur. In the aqueous phase, path B is the most feasible mechanism to account for the fact that the activation free energy of path B decreases compared with the corresponding path in the gas phase, whereas those of paths A and C increase. The main striking results are that the HSO3- group directly interacts with the C5=C6 bond rather than the N3=C4 bond and that the C5 methylation, compared with Cyt, by decreasing values of global electrophilicity index manifests that C5 methylation forms are less electrophilic power as well as by decreasing values of NPA charges on C5 site of the intermediates make the trend of addition reaction weaken, which is in agreement with the experimental observation that the rate of 5-MeCyt reaction is approximately 2 orders of magnitude slower than that of Cyt in the presence of bisulfite. Apart from cis and trans isomers, the rare third isomer where both the CH3 and SO3 occupy axial positions has been first found in the reactions of neutral and protonated 5-MeCyt with the HSO3- group. Furthermore, the transformation of the third isomer from the cis isomer can occur easily.