In the present study, we investigated the protective mechanism of paeoniflorin (PF), a monoterpene glycoside extracted from Radix Paeoniae alba roots, on MPP(+)-induced neurotoxicity in cultured rat pheochromocytoma cells (PC12). Our work included examination of cell viability assessment, amounts of released lactic dehydrogenase (LDH), intracellular Ca(2+) concentration, cell apoptosis, mitochondrial membrane potential, caspase-3 activity, and expression profiling of two apoptosis-related genes (Bcl-2 and Bax). It was shown that, PF functioned as an MPP(+) antagonist, being able to suppress apoptosis, decrease LDH release and Ca(2+) concentration, attenuate membrane potential collapse and, inhibit caspase-3 activation, decrease in Bax/Bcl-2 ratio. These observations suggest that PF could protect PC12 cells against MPP(+)-induced injury and the mechanism PF's neuroprotective effect was closely associated with Bcl-2 up-regulation and Bax down-regulation. PF has neuroprotective effects on MPP(+)-induced apoptosis in PC12 cells via regulating mitochondrial membrane potential and Bcl-2/Bax/caspase-3 signaling pathways, and this new insight will help develop a PF-based therapeutic strategy for treatmenting neurodegenerative diseases and injury.