Natural plants offer a treasure trove of resources for anticancer drug development. Clematis are widely used in Traditional Chinese Medicine. However, studies on the active substances in Clematis are scarce. In the present study, four monomer compounds were successfully extracted from this species and their inhibitory effects on the growth of breast cancer cells were investigated using bioactivity tests. Among them, Clematis hederagenin saponin (CHS) belongs to the class of triterpenoid saponins. CHS showed cytotoxic effects on breast cancer cells in a dose- and time-dependent manner. The compound also induced apoptosis in breast cancer cells in a time-dependent manner. Further investigation into the underlying mechanisms of apoptosis induction in breast cancer cells showed that the compound significantly reduced mitochondrial Apaf-1 and cytochrome c proteins in breast cancer cells. In addition, it upregulated the activities of caspase-3 and -9. In conclusion, CHS induced apoptosis in breast cancer cells through regulation of the mitochondrial apoptosis pathway. The results suggest that the hederagenin saponin extracted from Clematis ganpiniana offers great potential as a novel anti-breast cancer drug.