Glycogen synthase kinase-3β (GSK-3β) is closely involved in neuronal apoptosis and pathogenesis of many neurodegenerative diseases, such as Alzheimer's disease. However, whether GSK-3β mediates apoptosis of dopaminergic neurons in Parkinson's disease remains elusive. In this study, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism models, we investigated whether MPTP induces apoptosis of dopaminergic neurons through a GSK-3β-dependent pathway. MPTP caused a rapid activation of GSK-3β, evidenced by the decrease in level of phospho-Ser9 of GSK-3β and the increase in level of phospho-Ser396 of tau, a known GSK-3β substrate. Blockage of GSK-3β activity by its two specific inhibitors, indirubin-3′-oxime and AR-A014418, prevented dopaminergic neurons from MPTP-induced apoptosis. Additionally, inhibition of GSK-3β activity restored the depletion of striatal dopamine and ameliorated behavioral impairments caused by MPTP. These results indicate that GSK-3β is a critical intermediate of MPTP neurotoxicity, and inhibition of GSK-3β may provide a novel strategy to treat Parkinson's disease.