Indirubin-3-oxime (I3O), a synthetic derivative of indirubin, was originally designed as potent inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3β (GSK3β) for leukemia therapy. In the current study, we have shown, for the first time, that I3O prevented 6-hydroxydopamine (6OHDA)-induced neuronal apoptosis and intracellular reactive oxygen species accumulation in PC12 cells in a concentration-dependent manner. GSK3β inhibitors but not CDK5 inhibitors reduced the neurotoxicity induced by 6OHDA. Moreover, the activation of GSK3β was observed after 6OHDA treatment. Furthermore, 6OHDA substantially decreased the transcriptional activity of myocyte enhancer factor 2D (MEF2D), a transcription factor that plays an important role in dopaminergic neuron survival, and reduced nuclear localized MEF2D expression. Interestingly, indirubin-3-oxime and GSK3β inhibitors prevented 6OHDA-induced dysregulation of MEF2D. In addition, short hairpin RNA-mediated decrease of MEF2D expression significantly abolished the neuroprotective effects of indirubin-3-oxime. Collectively, our results strongly suggested that indirubin-3-oxime prevented 6OHDA-induced neurotoxicity via activating MEF2D, possibly through the inhibition of GSK3β. In view of the capability of indirubin-3-oxime to cross the blood–brain barrier, our findings further indicated that indirubin-3-oxime might be a novel drug candidate for neurodegenerative disorders, including Parkinson’s disease in particular.