Electron transport chain (ETC) dysfunction or hypoxia causes toxic NADH accumulation. How cells regenerate NAD⁺ under such conditions remains elusive. Here, integrating bioinformatic analysis and experimental validation, we identify glycerol-3-phosphate (Gro3P) biosynthesis as an endogenous NAD⁺-regeneration pathway. Under genetic or pharmacological ETC inhibition, disrupting Gro3P synthesis inhibits yeast proliferation, shortens lifespan of C. elegans, impairs growth of cancer cells in culture and in xenografts, and causes metabolic derangements in mouse liver. Moreover, the Gro3P shuttle selectively regenerates cytosolic NAD⁺ under mitochondrial complex I inhibition; enhancing Gro3P synthesis promotes shuttle activity to restore proliferation of complex I-impaired cells. Mouse brain has much lower levels of Gro3P synthesis enzymes as compared with other organs. Strikingly, enhancing Gro3P synthesis suppresses neuroinflammation and extends lifespan in the Ndufs4^−/− mice. Collectively, our results reveal Gro3P biosynthesis as an evolutionarily conserved coordinator of NADH/NAD⁺ redox homeostasis and present a therapeutic target for mitochondrial complex I diseases.