Prenatal diesel exhaust (DE) exposure is associated with detrimental health effects in offspring. Although previous reports suggest that DE exposure affects the brain of offspring in the developmental period, the molecular events associated with the health effects have largely remained unclear. We hypothesized that the DNA methylation state would be disrupted by prenatal DE exposure. In the present study, the authors examined the genome-wide DNA methylation state of the gene promoter and bioinformatically analyzed the obtained data to identify the molecular events related to disrupted DNA methylation. Pregnant C57BL/6J mice were exposed to DE (DEP; 0.1 mg/m³) in an inhalation chamber on gestational days 0-16. Brains were collected from 1-day-old and 21-day-old offspring. The genomewide DNA methylation state of the brain genome was analyzed by methylation-specific DNA immunoprecipitation and subsequent promoter tiling array analysis. The genes in which the DNA methylation level was affected by prenatal DE exposure were bioinformatically categorized using Gene Ontology (GO). Differentially methylated DNA regions were detected in all chromosomes in brains collected from both 1-day-old and 21-day-old offspring. Altered DNA methylation was observed independently of the presence of CpG island. Bioinformatic interpretation using GO terms showed that differentially methylated genes with CpG islands in their promoter were commonly enriched in neuronal differentiation and neurogenesis. The results suggest that prenatal DE exposure causes genome-wide disruption of DNA methylation in the brain. Disrupted DNA methylation would disturb neuronal development in the developmental period and may be associated with health and disease in later life.