Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease characterized by motor dysfunction like rigidity, rest tremor, and bradykinesia. A major pathological feature of PD is a loss of dopaminergic neurons in the striatum (ST) and substantia nigra (SN). Diosmetin is a flavonoid found primarily in citrus fruit, which is widely used to treat various diseases including cancer and ischemic stroke. We evaluated the neuroprotective effect of diosmetin on 1-methyl-4-phenyl-pyridinium (MPP⁺)-induced cytotoxicity in SH-SY5Y and PC12 cells. Diosmetin significantly protected neuronal cells against MPP⁺ in SH-SY5Y and PC12 cells. Next, we administered diosmetin to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-injected C57BL/6J mice and performed behavioral tests (grip, pole, and rotarod tests) to examine motor function. Diosmetin dramatically ameliorated motor dysfunction induced by MPTP in mice. Moreover, diosmetin significantly alleviated the dopaminergic neuronal loss in ST and SN of MPTP-treated mice. We also found that diosmetin recovered dopamine transporter loss from MPTP induced toxicity. Taken together, our results suggest that diosmetin would be a potential therapeutic candidate for PD.