Breast cancer progression is facilitated by the epithelial to mesenchymal transition (EMT), generating cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics. The fungus-derived sesterterpenoid natural produce compound, ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, although its molecular targets and mechanism of action are not well characterized. Herein, we utilized a model of mammary epithelial cells and breast cancer cell lines with and without EMT features to characterize the mechanism of selectivity towards EMT(+) cells by OpA. Proteins interacting with OpA in EMT(+) cells, including mitochondrial glutathione transporter SLC25A40, were identified through via mass spectrometry. We utilized trans-mitochondrial cybrids to determine that mitochondria mediate sensitivity to OpA. Furthermore, we report effects on glycolysis, oxidative metabolism, and disruption of metabolite abundance in the TCA cycle. Antioxidant mechanisms are activated by OpA in EMT(+) cells via the NRF2-ARE pathway, verified by decreased cytotoxicity in EMT(+) cells pretreated with the NRF2 activator CDDO. Collectively, we conclude that OpA selectivity toward EMT is mediated by the mitochondria, and at sub-cytotoxic levels, generates a metabolic shift leading to cell death countered by antioxidant mechanisms.