Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip resistance is a big threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress. In this study, DNA methylation map was developed for fall armyworm (FAW, Spodoptera frugiperda), and its function in regulating FAW Vip3Aa resistance was explored. FAW was screened by Vip3Aa for 10 generations, and bioassays indicated that Vip3Aa resistance increased trans-generationally. Based on the comparison of DNA methylation maps between Vip3Aa-resistant and -susceptible strains showed that gene body methylation was positively correlated with its expression. Moreover, the study demonstrated that a reduction in the methylation density within the gene body of a 3’5’-cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased resistance of FAW to Vip3Aa, which was validated through RNAi experiments. The mechanism of Vip3Aa resistance will improve the understanding of DNA methylation and its function in lepidoptera and provide a new perspective for making strategies to pest management.