The pathogenic mechanism of neurodegenerative brain disorder such as Alzheimer's disease (AD) has been still far from clearly understood. Previous research has identified that mitochondrial dysfunction induced by Aβ has been recognized as a hallmark in AD. Therefore, the effective agents targeting β-amyloid (Aβ)-induced mitochondrial dysfunction may be useful for the treatment or prevention of AD. In the present study, the neuroprotective effect of paeoniflorin (PF), one monoterpene glycoside isolated from the Chinese herb Radix Paeoniae alba, on Aβ25-35-induced toxicity in PC12 cells was investigated for the first time. The results showed that PF could attenuate or restore the cell injury induced by Aβ25-35 in PC12 cells through preventing mitochondrial dysfunction, including decreased mitochondrial membrane potential, increased cytochrome c release as well as activity of caspase-3 and caspase-9. Therefore, our data provide the evidence that PF could protect PC12 cells against Aβ25-35-induced neurotoxicity and might be a potentially therapeutic approach for AD in the future.