Elesclomol is a small-molecule investigational agent that selectively induces apoptosis in cancer cells by increasing oxidative stress. Elesclomol plus paclitaxel was shown to prolong progression-free survival compared with paclitaxel alone in a phase II clinical trial in patients with metastatic melanoma. However, the therapeutic potential of elesclomol in human breast cancer is unknown, and the signaling mechanism underlying the elesclomol effect is unclear. Here, we show that elesclomol alone modestly inhibited the growth of human breast cancer cells but not normal breast epithelial cells. Elesclomol potentiated doxorubicin- or paclitaxel-induced apoptosis and suppression of breast cancer cell growth. While both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were activated by elesclomol, elesclomol-induced apoptosis was only in part mediated by JNK1. The additive effect of elesclomol on chemotherapy drug-induced apoptosis was associated with increases in cleaved caspase-3, p21(Cip1), and p27(Kip1) and decreases in the Inhibitor of Apoptosis Protein levels and NF-kappaB activity. We also found that Akt/Hsp70 survival signaling was induced by elesclomol, which may reflect a cellular feedback mechanism. Blockade of Akt activation using a small-molecule inhibitor enhanced elesclomol-elicited apoptosis, while expression of a hyperactive Akt abolished the elesclomol effect. These data suggest that elesclomol's interaction with conventional chemotherapeutic and Akt-targeting agents may be exploited to induce apoptosis in breast cancer cells, and clinical trials of combined treatment of elesclomol and chemotherapy drugs or Akt-targeting agents in breast cancer patients, especially the estrogen receptor negative subgroup, may be warranted.