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Objective(s): Widely used Titanium dioxide nanoparticles (TiO2) enter into the body and cause various organ damages. Therefore, we aimed to study the effect of TiO2 on the substantia nigra of midbrain. Materials and Methods: 40 male BALB/c mice were randomly divided into five groups: three groups received TiO2 at doses of 10, 25, and 50 mg/kg, the fourth group received normal saline for 45 days by gavage, and control group (without intervention). Then, Motor tests including pole and hanging tests were done to investigate motor disorders. The animal brain was removed for histological purposes. Accordingly, immunohistochemistry was performed to detect tyrosine hydroxylase positive cells, and then toluidine blue staining was done to identify dark neurons in the substantia nigra. Eventually, the total number of these neurons were counted using stereological methods in different groups.Results: The results showed that the time recorded for mice to turn completely downward on the pole in the TiO2-50 group increased and also the time recorded for animals to hang on the wire in the hanging test significantly decreased (P