Abstract: The present study is to investigate the protective actions of guggulsterone against the cytotoxicity produced by exposure to hydrogen peroxide (H₂O₂) in PC12 cells. It was evaluated by MTT ［3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide］ reduction assay, lactate dehydrogenase (LDH) release assay, and the release of nitric oxide (NO). ROS and Ca²⁺ in cells were evaluated by DCFH and Fura 2-AM, respectively. Mitochondrial membrane potential (MMP) was assessed by the retention of rhodamine 123 (Rh 123). Apoptosis and morphological alteration in PC12 cells were monitored with flow cytometry and electric microscope. Vitamin E, a potent antioxidant, was employed as a comparative agent. The results showed that preincubation of PC12 cells with guggulsterone (0.1-10 μmol·L^-1) prevented cytotoxicity induced by H₂O₂. Extracellular accumulation of LDH, NO and intracellular accumulation of ROS, Ca²⁺ resulting from H₂O₂ were significantly reduced by guggulsterone. Incubation of cells with H₂O₂ caused a marked decrease in MMP, which was significantly inhibited by guggulsterone. The percentage of H₂O₂-induced apoptosis in PC12 cells was 24.3%, and decreased in the presence of guggulsterone (0.1-10 μmol·L^-1) by 18.4%, 15.9%, 11.8%, respectively. Guggulsterone exhibited comparable potency against oxidative stress induced by H₂O₂ in PC12 cells as that of vitamin E. The present findings showed that guggulsterone attenuated H₂O₂-induced cytotoxicity, extracellular accumulation of LDH and NO, intracellular accumulation of ROS and Ca²⁺, loss of MMP, and apoptosis, which may represent the cellular mechanisms for its neuroprotective action.