Brain iron level is significantly increased in the substantia nigra (SN) of Parkinson's disease (PD) patients. It is unclear whether iron overload causes dopaminergic neuronal death in the SN of PD or iron deposition is a byproduct of PD. In this study, two mouse models with high levels of brain iron were used, ceruloplasmin knockout (CP-/-) mice and mice receiving an intracerebroventricular injection of ferric citrate (FAC). After intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the dopaminergic neuron damage in the substantia nigra pars compacta (SNpc) of the mice and their behavior were assessed. Our ﬁndings suggest that the injection of FAC or the absence of the CP gene may exacerbate both apoptosis in TH-positive neurons and behavioral symptoms in mice after MPTP treatment. Intracerebroventricular injection of desferrioxamine in CP knockout mice, however, may significantly alleviate the neuronal damage induced by MPTP. Furthermore, our findings suggest that increased nigral iron content exacerbates oxidative stress levels in the brain, promoting cell apoptosis through the Bcl-2/Bax pathway and the activated caspase-3 pathway. Therefore, these results indicate that brain iron overload exacerbates the dopaminergic neuronal death and initiates the onset of PD.