The cyclobutane pyrimidine dimer (CPD) is a major photoproduct of deoxyribonucleic acid (DNA) that is damaged by ultraviolet light. This DNA lesion can be repaired by DNA photolyase with the aid of UV light and two cofactors. To understand the repair mechanism of CPD and whether protonation of CPD participates in the DNA repair process, the cycloreversion reactions of four CPD models and proton transfers between the adjacent residue Glu283 and CPD models were explored through the quantum mechanical method. Two-dimensional maps of potential energy surface in a vacuum and in implicit water solution were calculated at the ωB97XD/6-311++G(2df,2pd) level. One-dimensional potential energy profiles were computed for proton transfer reactions. Among the models that have been considered, both in a vacuum and in water solution, the results indicate that the most likely repair mechanism involves CPD•2- radical anion splitting in a stepwise manner. C5-C5' splits first, and C6-C6' splits later. The computed free energies of activation of the two splitting steps are 0.9 and 3.1 kcal/mol, respectively. The adjacent Glu283 may stabilize the CPD•2- radical anion through hydrogen bond and increase the quantum yield; however, protonating the CPD radical anion by Glu283 cannot accelerate the rate of ring opening.