One million females are diagnosed worldwide every year with breast cancer, and the mortality rate of these patients remains high. Several treatments, including surgery, are available for breast cancer. β‑Lapachone (β‑Lap), a natural quinone compound, has been developed for cancer treatment due to its strong cytotoxic effect through its action on NAD(P)H:quinone oxidoreductase 1 (NQO1)‑dependent activity. However, the mechanism in regards to how β‑Lap induces cytotoxicity in breast cancer cells is still elusive. In the present study, we showed that β‑Lap induced apoptotic cell death via activation of protein kinase A (PKA) in NQO1‑overexpressing MDA‑MB‑231 human breast cancer cells. This PKA‑dependent cell death was observed solely in NQO1‑overexpressing 231 cells via the high production of reactive oxygen species (ROS). Cell survival of antioxidant [N‑acetylcysteine (NAC)]‑treated NQO1‑overexpressing 231 cells was significantly recovered, and NQO1‑negative 231 cells did not respond to β‑Lap. Antiapoptotic proteins such as Bcl2 and Bcl‑xL were decreased, while proapoptotic proteins, including cytochrome c, activation of caspase‑3, and cleavage of PARP were increased after β‑Lap treatment of NQO1‑overexpressing 231 cells. Furthermore, PKA activators, forskolin or dibutyryl‑cAMP, an analog of cAMP, aggravated the β‑Lap‑induced apoptotic cell death by decreasing antiapoptotic proteins and further activating proapoptotic proteins in NQO1‑positive 231 cells. Treatment with a PKA inhibiter, H89, significantly increased cell viability even in NQO1‑overexpressing cells treated with β‑Lap. These data showed that β‑Lap activated PKA via ROS accumulation, subsequently leading to apoptotic cell death in NQO1‑positive breast cancer cells.