There is an increasing body of evidence suggesting that a single exposure to certain chemicals can have transgenerational effects, with the underlying mechanism believed to be epigenetic. However, it remains largely unknown whether psychiatric conditions like ADHD or autism, induced by environmental chemicals, can be inherited across generations. Pregnant rats were purchased from a commercial breeder. On the 7th day of gestation (E7), they were divided into two groups: one group was orally exposed to silver nanoparticles (AgNP; 4 mg/kg), while the control group received vehicle alone. The subsequent generation (F1) underwent spontaneous motor activity (SMA) measurements at 8-11 weeks of age. For breeding at 26 weeks of age, rats with higher SMA were selected from hyperactive litters, while untreated rats were randomly selected. This process was continued for four generations in both groups. The AgNP-primed rats at 4th generation displayed significantly higher SMA, 1.8 times greater than that of untreated rats. Intraperitoneal injection of valproic acid (150 mg/kg), an epigenetic modifier to 5-day-old rats causes adult hyperactivity (1.4-fold), suggesting that epigenetic modification contributes to rat hyperactivity. Global DNA methylation profiles in the mesencephalon were positively correlated in both groups of hyperactive rats. Furthermore, there were 7-8 common genes showing both hypermethylation and hypomethylation, which are involved in neuronal development, neuronal function, transcriptional activity, DNA binding activity, cell differentiation, ubiquitination processes, or histone modification, including Pax 6 and Mecp 2. Thus, it is most likely that rats retain hyperactivity through mesencephalic DNA methylation status across transgeneration.