Glucose metabolism has been studied extensively, but the role of glucose-derived excretory glycerol remains unclear. Here, we show that hypoxia induces NADH accumulation to promote glycerol excretion and this pathway consumes NADH continuously, thus attenuating its accumulation and reductive stress. Aldolase B (ALDOB) accounts for glycerol biosynthesis by forming a complex with glycerol-3-phosphate dehydrogenases, GPD1 and GPD1L. Blocking GPD1, GPD1L, or glycerol 3-phosphate phosphatase exacerbates reductive stress and suppresses cell proliferation under hypoxia and tumor growth in vivo. Over-expression of these enzymes increases glycerol excretion but still reduces cell viability under hypoxia and tumor proliferation due to energy stress. AMPK inactivates ALDOB to mitigate glycerol synthesis that dissipates ATP, alleviating NADH accumulation-induced energy crisis. Therefore, glycerol biosynthesis/excretion regulates the trade-off between reductive stress and energy stress. Moreover, this mode of regulation seems to be prevalent in reductive stress-driven transformations, enhancing our understanding of the metabolic complexity and guiding tumor treatment.