Accumulating evidence suggest mitochondria-mediated pathways play an important role in dopaminergic neuronal cell death in Parkinson's disease (PD). Drp1, a key regulator of mitochondrial fission, has been shown to be activated and translocated to mitochondria under stress, leading to excessive mitochondria fission and dopaminergic neuronal death in vitro. However, whether Drp1 inhibition can lead to long term stable preservation of dopaminergic neurons in PD-related mouse models remains unknown. In this study, using a classical MPTP animal PD model, we showed for the first time Drp1 activation and mitochondrial translocation in vivo after MPTP administration. Inhibition of Drp1 activation by a selective peptide inhibitor P110, blocked MPTP-induced Drp1 mitochondrial translocation and attenuated dopaminergic neuronal loss, dopaminergic nerve terminal damage and behavioral deficits caused by MPTP. MPTP-induced microglial activation and astrogliosis were not affected by P110 treatment. Instead, inhibition of Drp1 mitochondrial translocation diminished MPTP-induced p53, BAX and PUMA mitochondrial translocation. This study demonstrates that inhibition of Drp1 hyperactivation by a Drp1 peptide inhibitor P110 is neuroprotective in a MPTP animal model. Our data also suggest that the protective effects of P110 treatment might be mediated by inhibiting the p53 mediated apoptotic pathways in neurons through inhibition of Drp1-dependent p53 mitochondrial translocation.