Abstract: In order to accurately evaluate the activity of DNA methyltransferase (DNMT) on the amount of O⁶-methylguanine (O⁶-mG) produced by α-methylene hydroxylation metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to determine the activity of DNMT, the level of DNA methylation, the contents of 4-oxo-4-(3-pyridyl)butyric acid (OPBA), 4-hydroxy-4-(3-pyridyl)butyric acid (HPBA) and O⁶-mG in the livers and lungs of mice in physiological saline-treated group and decitabine-treated group after 100 mg/kg NNK administration separately. The results showed that:1) Decitabine inhibited the activity of DNA methyltransferase and decreased the level of DNA methylation. 2) Decitabine did not significantly affect the contents of OPBA and HPBA produced by α-methylene hydroxylation metabolism of NNK. 3) The amount of O⁶-mG produced through α-methylene hydroxylation metabolism of NNK could be reduced by inhibiting the activity of DNA methyltransferase.