In the development and progression of Alzheimer's disease (AD), β-amyloid peptide (Aβ) that induced cytotoxicity containing apoptosis and excess production of reactive oxygen species (ROS) is considered as a causal role. The aim of present study is to investigate the protective effect of Trihexyphenidyl (THY) on Aβ(25-35)-induced cytotoxicity in cultured rat pheochromocytoma (PC12) cells. In this report, the cell survival was measured by MTT assay, the enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), the contents of lipid peroxidation products malondialdehyde (MDA) and ROS in the cells were determined. Acridine orange (AO) was used to observe the morphological characteristic of apoptotic cells. Mitochondrial membrane potential in PC12 cells were monitored by fluorospectrophotometer combining with Rh123. As a cell permeable fluorescent probe, Fura-2/AM was employed to detect intracellular [Ca(2+)]. The results showed that after incubation with Aβ(25-35) (10 μM) for 24 h, there were decreased changes in cell viability, SOD, and GSH-PX activity as well as mitochondrial membrane potential, in contrast, the levels of [Ca(2+)](i), ROS, and MDA were increased, THY significantly attenuated all the changes induced by Aβ(25-35), indicating that THY exhibited protective effect against Aβ(25-35)-induced cytotoxicity, which may represent the cellular mechanisms of the action.