Abstract: The hydrolysis reaction mechanism and potential energy surface of 5-methylcytosine (m^5C) were investigated using density functional theory (DFT) at the B3LYP/6-31G^** level. The results show that the hydrolysis reaction of m^5C includes two pathways: (A)With the water attacking at m^5C, thymine is formed; (B) This pathway is associated with a tetracoordinated intermediate formed and decomposed. Because the hydrolysis reaction of m^5C has a higher barrier, the hydrolysis reaction of m^5C does not take place for the opening system, which is agreement with the experimental result. The present calculations have rationalized and verified all the possible reaction channels