Diesel exhaust (DE) is one of the major air pollutants in the world. DE disrupts steroid hormone levels, which may result from the disruption of spermatogenesis. Steroidogenesis occurs not only in the testis but also in the brain. Therefore, we investigated the effects of nanoparticle-rich DE (NR-DE) on steroidogenesis in both the testis and hippocampus. Exposure to NR-DE at concentrations comparable to the environmental standard for particulate matters 2.5 (PM2.5) in Japan increased plasma testosterone level. This exposure increased the expression levels of genes involved in steroidogenesis in the testis, but not in the hippocampus, suggesting that NR-DE disrupts steroid hormone balance. This finding suggests the need to reconsider the environmental limit of PM2.5 in Japan.