The mechanism for the deamination reaction of 5-methylcytosine with H2O in protic medium was investigated using DFT calculations at the B3LYP/6-311G(d,p) level of theory. Two pathways were found. Pathway 5mA is a two-step mechanism where the N3-protonated 5-MeCyt undergoes a nucleophilic attack to carbon C4 by a water dimer before the elimination of an ammonium cation. Pathway 5mB is a three-step mechanism where neutral 5-MeCyt is directly attacked by a water dimer. The resulting intermediate is then protonated to allow the elimination of an ammonium cation. Both pathways lead to the formation of thymine in interaction with an ammonium cation and a water molecule. Pathway 5mA can explain the spontaneous deamination of 5-MeCyt in protic medium at acidic pH, whereas pathway 5mB is more representative of the deamination in protic medium at neutral pH. The nucleophilic addition of the water dimer is rate-determining in both pathways and is associated with an activation free energy in aqueous solution of 137.4 kJ/mol for pathway 5mA and 134.1 kJ/mol for pathway 5mB. This latter value is in agreement with the experimental observation that 5-MeCyt deaminates four- to fivefold faster than Cyt at neutral pH. Both electrostatic and electron-transfer contributions appear to have significant importance. In vacuum, the former one dominates when the substrate is positively charged and the latter one when it is neutral.