Alpha-synuclein (α-Syn) accumulation is significantly correlated with motor deficits and mitochondrial dysfunction in Parkinson's disease (PD), but the molecular mechanism underlying its pathogenesis is unclear. In this study, we investigated the effects of treadmill exercise on motor deficits and mitochondrial dysfunction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treadmill exercise inhibited dopaminergic neuron loss by promoting the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) and seemed to improve cell survival by reducing α-Syn expression. Most importantly, treadmill exercise increased expression of the mitochondrial import machinery proteins TOM-40, TOM-20, and TIM-23. This was associated with decreased α-Syn expression and subsequent upregulation of the mitochondrial proteins COX-I, COX-IV, and mtHSP70. Taken together, these results indicate that treadmill exercise may ameliorate motor deficits and improve mitochondrial dysfunction by reducing α-Syn expression in the MPTP-induced mouse model of PD.