Combined quantum-mechanical/molecular-mechanical (QM/MM) approaches have been applied to investigate the detailed reaction mechanism of human O-6-alkylguanine-DNA alkyltransferase (ACT). ACT is a direct DNA repair protein that is capable of repairing alkylated DNA by transferring the methyl group to the thiol group of a cysteine residue (Cys145) in the active site in an irreversible and stoichiometric reaction. Our QM/MM calculations reveal that the methyl group transferring step is expected to occur through two steps, in which the methyl carbocation generating step is the rate-determining step with an energy barrier of 14.4 kcal/mol at the QM/MM B3LYP/6-31G(d,p)//CHARMM22 level of theory. It is different from the previous theoretical studies based on QM calculations by using a cluster model in which the methyl group transferring step is a one-step process with a higher energy barrier.