Acetamiprid, which is a neonicotinoid insecticide, is used to control leafy vegetables, fruiting vegetables, fir seeds, citrus fruits, pome fruits, grapes, cotton and ornamental plants and absorbent insects on flowers. The present study aim to evaluate global DNA methylation and gene expression of DNA methylation related enzymes in liver and brain tissues of male Sprague-Dawley rats after a 90-day subchronic exposure to acetamiprid at low doses of 12.5, 25 and 35 mg/kg body weight (b.w.). Global DNA methylation resulted in a significant decrease in the levels of 5-methylcytosine (5-mC%) at the doses of 25 and 35 mg/kg b.w. in the liver and 35 mg/kg b.w. in the brain compared to the vehicle control group. Consistently, expression of DNA methyltransferase enzymes decreased at doses of 12.5, 25 and 35 mg/kg b.w. in liver and 35 mg/kg b.w. in brain. It has been suggested that non-genotoxic (epigenetic) mechanisms may be involved in the toxicity of acetamiprid and further investigations are needed to elucidate the epigenetic effects of neonicotinoid insecticides.