<p>Targeting mitochondrial metabolism represents a novel approach in the discovery and development of anti-tumor drugs. Celastrol (Cel) is a naturally-derived small molecule from <i>Tripterygium wilfordii</i> with significant anticancer activities. In this study, we investigated the anti-tumor mechanisms involving mitochondrial metabolic reprogramming regulated by Cel in breast cancer (BRCA). We showed that Cel potently inhibited the proliferation of triple-negative breast cancer cells (MDA-MB-231) and ER⁺ breast cancer cells (MCF-7) with IC₅₀ values of 2.15 μM and 2.29 μM, respectively. Administration of Cel (5, 2, 2 mg/kg, i.p. for three times after tumor formation) significantly suppressed the tumor growth in syngeneic allograft and CDX breast cancer mouse models. Using activity-based protein profiling (ABPP) technology, we identified mitochondrial isocitrate dehydrogenases (including IDH2 and IDH3A, collectively referred to as mito-IDHs) as direct targets of Cel. We demonstrated that Cel significantly inhibited mito-IDHs mediated mitochondrial metabolism to induce the accumulation of metabolites α-ketoglutaric acid, and that Cel enhanced the interaction between DPYSL2 with IDH3A while promoting the accumulation of DPYSL2 within mitochondria of BRCA cells resulting in inactivation of JAK/STAT pathway and ultimately induced ferroptosis and apoptosis in cancer cells. Collectively, this study elucidates a pharmacological mechanism by which Cel exerts its tumor-inhibiting effects through modulation of mitochondrial metabolism. Furthermore, it provides compelling evidence supporting Cel as a promising candidate for development as a small-molecule inhibitor targeting mitochondrial metabolism.</p><p></p>