Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy.