Postharvest decay, primarily caused by pathogenic fungi in ripening fruits and fresh vegetables, poses a challenge to agricultural sustainability and results in significant economic losses. The regulation of the fruit ripening by DNA methylation has been well demonstrated, while defense response of fruit underlying epigenetic regulation against postharvest decay remain uncertain. In the present study, treatment of tomato fruits with the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) notably decreased their susceptibility to gray mold. Following 5-Aza treatment, we observed a substantial increase in activities of chitinase (CHI) and glucanase (GLU) in tomato fruits, as well as an increase in the expression of the dicer like SlDCL2 gene family. Suppression of SlDCL2c through double-stranded RNA-induced RNA interference (RNAi) resulted in a decrease in the expression of chitinases CHI3, CHI9, Class V chitinase, and endochitinase 4 by 71%, 29%, 55%, 64%, as well as glucanases Cel1, Cel2, and GluB by 19%, 93%, and 87%, respectively. This was accompanied by decreased activities of resistance-related enzymes, including CHI and GLU. The expression levels of genes phenylalanine ammonia-lyase PAL2, peroxidase POD 12, POD P7, CCR1, CYP84A2, and COMT in phenylpropanoid biosynthesis pathway also decreased by 33%, 53%, 18%, 50%, 30%, and 24% in SlDCL2c-RNAi fruit, resulting in decreased activities of PAL and POD. Consequently, the lesion diameter of gray mold in SlDCL2c-RNAi fruit increased by 55% compared to the control group. Overall, the present study indicated that DNA methyltransferase inhibitor 5-Aza reduces susceptibility of tomato fruit to gray mold through regulation of DCL2c-mediated inducible defense response.