Alzheimer's disease (AD) is the most common form of dementia. Oxidative stress is one of the earliest events in the neurological and pathological changes of AD. L-3-n-butyl-phthalide (L-NBP), an anti-cerebral ischemia agent, has been shown a potential in AD treatment. In this study, we investigated the neuroprotective effect of L-NBP on hydrogen peroxide (H₂O₂)-induced apoptosis in human neuroblastoma SK-N-SH cells. H₂O₂ significantly reduced cell viability and increased the number of apoptotic-like cells, indicating that H₂O₂ induced neurotoxicity. In addition, real-time PCR and western blot studies showed that Bcl-2 and Bcl-w expressions were decreased, and Bax expression was increased with H₂O₂ treatment. Moreover, protein kinase C (PKC) α expression was down-regulated after H₂O₂ treatment. All of these phenotypes induced by H₂O₂ were markedly reversed by L-NBP. Pretreatment with L-NBP significantly increased cell viability of H₂O₂-damaged cells, and reduced H₂O₂-induced neuronal apoptosis. L-NBP treatment at dose of 10 μM inhibited H₂O₂-induced down-regulation of Bcl-2, Bcl-w, and PKCα but also attenuated the overexpression of Bax. PKC inhibitor, calphostin C, significantly attenuated the protective effects of L-NBP. Our findings suggest that L-NBP may protect neurons against H₂O₂-induced apoptosis by modulating apoptosis-related genes and activating PKCα pathway.