20-hydroxyecdysone (20E), a steroidal prohormone, is secreted from the prothoracic glands. While 20E has been shown to have neuroprotective effects in Parkinson's disease (PD) models in vitro, its effects have not yet been examined in vivo. We sought to assess the behavioral and mechanistic effects of 20E on MPTP-induced toxicity in mice. To this end, we used behavioral tests, stereological analyses of dopaminergic neurons by tyrosine hydroxylase immunohistochemistry, and assessments of apoptotic mechanisms, focusing on Nrf2 signaling through Western blotting and ELISA assays. A 20E treatment protected against MPTP-induced motor incoordination, postural imbalance, and bradykinesia, and significantly reduced dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) and the striatum (ST). It also attenuated dopamine deficiency in the ST, modulated levels of antioxidative enzymes superoxide dismutase, catalase, and glutathione in the SNpc, increased the Bcl-2/Bax ratio, and inhibited cytosolic cytochrome c release and caspase-9, -7, and -3 activity in the SNpc. These results indicated that 20E inhibited the apoptotic cascade. Furthermore, the attenuation of MPTP neurotoxicity was associated with inhibited cleaved-caspase signaling pathways, along with upregulated Nrf2 pathways in the SNpc, suggesting that 20E mitigates MPTP-induced neurotoxicity via mitochondria-mediated apoptosis by modulating anti-oxidative activities. Our results suggest that 20E can inhibit MPTP-induced behavioral and neurotoxic effects in mice. This lays the foundation for further research on 20E as a potential target for therapeutic use. Display omitted •The effects of 20-hydroxyecdysone in Parkinson's disease were assessed.•20E can inhibit MPTP-induced behavioral and neurotoxic effects.•20E may be a potential therapeutic target.