Emerging evidence suggests that alpha-synuclein (alpha-syn), which is traditionally thought to have a pathophysiological role in neurodegenerative diseases, can have neuroprotective effects. This study aimed to investigate whether endogenous alpha-syn in neurons can be induced by valproic acid (VPA), a mood-stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, and if so, whether the alpha-syn induction is neuroprotective. VPA treatment of rat cerebellar granule cells caused a robust dose- and time-dependent increase in levels of alpha-syn protein and mRNA and in the intensity of alpha-syn immunostaining. Knockdown of VPA-induced alpha-syn overexpression with alpha-syn antisense oligonucleotides or siRNA completely blocked VPA-induced neuroprotection. alpha-Syn knockdown also exacerbated glutamate neurotoxicity, stimulated the expression of the proapoptotic gene ubiquitin-conjugating enzyme E2N, and downregulated the expression of the anti-apoptotic gene Bcl-2. Induction of alpha-syn by VPA was associated with inhibition of HDAC activity, resulting in hyperacetylation of histone H3 in the alpha-syn promoter and a marked increase in alpha-syn promoter activity. Moreover, VPA-induced alpha-syn induction and neuroprotection were mimicked by HDAC inhibitors sodium 4-phenylbutyrate and trichostatin A (TSA). alpha-syn was also induced by VPA in rat cerebral cortical neurons. Additionally, treatment of rats with VPA, sodium butyrate, or TSA markedly increased alpha-syn protein levels in the cortex and cerebellum. Together, our results demonstrate for the first time that VPA induces alpha-syn in neurons through inhibition of HDAC and that this alpha-syn induction is critically involved in neuroprotection against glutamate excitotoxicity. Clinically, VPA may represent a suitable treatment for excitotoxicity-related neurodegenerative diseases.