A previous study showed that single intracisternal administration of p-nitrotoluene into neonatal rats caused hyperactivity. To evaluate the neural risk assessment of p-nitrotoluene, it is crucial to test the potential of the chemical via environmental exposure route. In this study, we tested the hypothesis that oral exposure to p-nitrotoluene would exhibit the effects observed previously with direct instillation. Oral administration of 600 μg/day p-nitrotoluene into male Wistar rat pups, 5 days to 3 weeks of age, caused significant hyperactivity at 4-5 weeks of age. Treated rats were about 1.3 times active in the nocturnal phase than the vehicle-treated control rats (p < 0.05). The long-term effects of the chemical caused a large reduction in tyrosine hydroxylase (TH) immunoreactivity in the midbrain at 7 weeks of age, at which terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling-positive cells were detected. Immunohistochemical analysis of glial fibrillary acidic protein also revealed p-nitrotoluene-induced gliosis in the substantia nigra, suggesting neurodegeneration. Furthermore, neonatal p-nitrotoluene-induced lesion decreased the gene expression levels of dopamine transporter in adult rats. Thus, we conclude that p-nitrotoluene via oral exposure route caused rat hyperactivity, concomitant with gliosis and impairment of TH immunoreactivity, most likely due to degeneration of dopaminergic neurons.