Mitotic assembly of respiratory supercomplexes ensures accurate cell division and fuels tumor growth.

As cells progress from interphase into mitosis, fluctuating metabolic demands coincide with mitochondrial fission. However, the mechanisms by which mitochondria coordinate morphological changes and metabolic adjustments during mitosis remain poorly understood. Using proteomic analysis of BN-PAGE fractions, we show that assembly of the mitochondrial respiratory supercomplexes, comprising electron transport chain complexes I, III, and IV, is markedly enhanced during mitosis in HeLa and MDA-MB-468 cancer cells. Mechanistically, the upregulation of specific CI subunits, including NDUFA3, drives the modular assembly of supercomplexes in mitosis. We further demonstrate that CDK1 promotes the translation of NDUFA3 to enable supercomplex formation. Disruption of this process impairs mitochondrial integrity, energy production, and redox homeostasis, leading to ROS accumulation that triggers mitotic cell death and chromosome segregation defects across multiple models. Importantly, inhibiting mitotic supercomplex assembly induces chromosome mis‑segregation and suppresses tumor growth in vivo. Our findings reveal supercomplex assembly as a key mechanism coordinating mitochondrial fission with metabolic adaptation to support cell division and tumor proliferation in these cancer cell contexts.