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Highlights • Azacitidine (5-azaC) could promote the early ripening of the grape. • The treatment of 100 μM 5-azaC reduced the level of DNA methylation during the development of 'Kyoho' grape berries. • The ROS metabolic activities and related physiological indexes in berries treated with 5-azaC were also investigated. • There was a connection between ROS metabolism and DNA methylation during the grape berry development. Abstract DNA methylation has been proved to function in fruit ripening. The different concentrations of azacitidine (5-azaC), an inhibitor of methyltransferase, were sprayed on 'Kyoho' grape berries to explore the changes of DNA methylation, the effect of 5-azaC on berry ripening, and the relationship between DNA methylation and reactive oxygen species (ROS) metabolism during the grape berry development. The results showed that the treatment of 100 μM 5-azaC could promote the berry ripening of 'Kyoho' 20 days earlier than the control. The assessment of methylation by methylation sensitive amplification polymorphism (MSAP) markers after the treatment of 100 μM 5-azaC, demonstrated that the methylation rate of the treatment was all lower than the control at any berry development periods. 5-azaC treatments influenced the ROS metabolic activities and related physiological indexes of berries. The sucrose and soluble solid content of 5-azaC-treated berries were significantly higher than control at 75 dpa. The change trend of H 2 O 2 content of the control was similar with the 100 μM 5-azaC treatment. All treatments enhanced rates of superoxide anion (O 2 −) production. Superoxide dismutase (SOD) activity in treated berries were higher than the control and had significant difference with the control at 35 dpa. The results demonstrated that 5-azaC could accelerate grape berries ripening, and there was a connection between ROS metabolism and DNA methylation during the grape berry development. [ABSTRACT FROM AUTHOR]