To investigate the protective effect and the underlying mechanism of polygalasaponin F (PS-F) against rotenone-induced PC12 cells, the cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The cell apoptosis rate was analyzed using flow cytometry. The reactive oxygen species was examined using 2',7'-dichlorofluorescin diacetate, and the adenosine triphosphate depletion was examined using a luciferase-coupled quantification assay. JC-1 staining was used to detect the mitochondrial membrane potential. Western blotting analysis was used to determine cytochrome c, p53, Bax, Bcl-2, and caspase-3. Treatment of PC12 cells with rotenone (1-10 μmol/l) significantly reduced the cell viability in a concentration-dependent manner. Treatment with PS-F (0.1, 1, and 10 μmol/l) increased the viability of rotenone-induced PC12 cells, decreased rotenone-induced apoptosis, restored rotenone-induced mitochondrial dysfunction, and suppressed rotenone-induced protein expression. PS-F showed a dose-dependent manner in all the treatments. PS-F protects PC12 cells against rotenone-induced apoptosis via ameliorating the mitochondrial dysfunction. Thus, PS-F may be a potential bioactive compound for the treatment of Parkinson's disease.