Valproate (VPA) is a commonly prescribed mood stabilizer. However, emerging evidence indicates that VPA administration may cause reversible symptoms of Parkinsonism and cognitive decline (P/CD) in some manic patients. The mechanism of this phenomenon is unknown. In this study, we used human SY5Y neuroblastoma cells as a neuronal model to investigate the effects of VPA on neurite outgrowth and neurofilament expression. Data showed that the treatment with VPA at therapeutic plasma levels (0.5 mM) significantly reduced cell proliferation from day 4 through day 6, and neurite outgrowth length from day 1 through day 6. Conversely, VPA had no effect on cell proliferation of human CCF astrocytoma cells but stimulated nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 pheochromocytoma cells. In parallel to these alterations in human SY5Y cells, both mRNA and protein levels of neurofilament 160 (NF160) were significantly reduced, starting at day 2 and day 3, respectively, by the treatment. The inhibition of neurite outgrowth by VPA was completely reversed 2 days after cessation of VPA exposure. Furthermore, NF160 protein levels also rebounded to control levels after VPA removal. NGF application significantly alleviated the inhibition of neurite outgrowth by VPA. These data suggest that VPA-modulated NF160 expression was involved in the inhibition and the reversal of neurite outgrowth in human neuronal cells.