25 Increasing evidence indicates that insecticides induce various diseases via DNA 26 methylation. DNA methylation plays an important role during cell differentiation and 27 exhibits its greatest vulnerability to environmental factors during embryogenesis. 28 Therefore, it is important to evaluate the effects on DNA methylation at the early stage of 29 cell differentiation to understand developmental toxicity. However, DNA methylation 30 induced by insecticides and the associated effects on cell differentiation are unclear. In 31 this research, we introduced a high-content approach utilizing mouse embryonic stem 32 cells harboring enhanced green fluorescent protein fused with methyl CpG-binding 33 protein to evaluate global DNA methylation induced by various insecticides. DNA 34 methylation was assessed in 22 genes after pesticide exposure to investigate the 35 relationships with biological processes such as cell cycle, cell apoptosis, and cell 36 differentiation. Exposure to acetamiprid, imidacloprid, carbaryl, and o,p′-DDT increased 37 the granular intensity, indicating their global DNA-methylating effects. Exposure to 38 imidacloprid decreased DNA methylation in genes such as Cdkn2a, Dapk1, Cdh1, Mlh1, 39 Timp3, and Rarb, indicating the potential influence of the DNA methylation pattern on 40 cell differentiation. We developed a promising approach for evaluating global DNA 41 methylation, and our findings suggested that imidacloprid might exhibit developmental 42 effects through DNA methylation pattern. 43 44 45 46 47 48