Proteomic analysis of 13 bands from BN-PAGE immunostaining of mouse skeletal muscle mitochondrial proteins: Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce the Q; however, in eukaryotes, only the OXPHOS-complex III (CIII) oxidizes the QH2. The mechanism of action of CIII is still debated. We demonstrate that the Q-reductase ETFDH is essential for CIII activity in skeletal muscle. We identify a complex involving ETFDH, CIII, and the Q-biosynthesis regulator COQ2 that directs electrons from lipid substrates to the respiratory chain, reducing electron leak and ROS production. This metabolon maintains Q levels, minimizes QH2-reductive stress, and improves OXPHOS efficiency. Muscle-specific ETFDH-/- mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine