Brucine, a weak alkaline indole alkaloid, is one of the main bioactive and toxic constituents of Strychnos nux-vomica L., which exerts multiple pharmacological activities, such as anti-tumor, anti-inflammatory, and analgesic effect. However, its potential toxic effects limited its clinical application, especially central nervous system toxicity. The present study was designed to investigate the neurotoxicity and mechanism of brucine. Our results showed that brucine significantly induced Neuro-2a cells and primary astrocyte death, as evidenced by MTT assay and LDH release. Moreover, transcriptome analysis indicated that PPAR/NF-kappa B and apoptosis signaling pathways were involved in the brucine-induced cytotoxicity in Neuro-2a cells. Subsequently, in fact, brucine evidently inhibited PPAR gamma and promoted phosphorylation of NF-kappa B. Furthermore, PPAR gamma inhibitor aggravated the neurotoxicity, while NF-kappa B inhibitor substantially reversed brucine-induced neurotoxicity. Moreover, brucine also significantly induced neuronal apoptosis and triggered increase in ratio of Bax/Bcl-2 and level of cleaved caspase 3, as well as its activity as evidenced by TUNEL staining and Western blot. Furthermore, molecular docking analysis predicted that brucine directly bound to caspase 3. Intriguingly, a caspase 3 inhibitor (Z-DEVE-FMK) largely abolished the neurotoxicity of brucine. Our results reveal that brucine-induced neurotoxicity via activation of PPAR gamma/NF-kappa B/caspase 3-dependent apoptosis pathway. These findings will provide a novel strategy against brucine-induced neurotoxicity.