AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress AMPK is a key regulator of metabolic adaptation, particularly under energy stress conditions that lower intracellular ATP levels. This study shows that AMPK activation during energy stress prolongs cell survival by regulating redox balance. Under these conditions, NADPH generation via the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. AMPK inhibits the acetyl-CoA carboxylases ACC1 and ACC2, which reduces NADPH consumption in fatty-acid synthesis and increases NADPH generation through fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas activation of ACC1 or ACC2 attenuates these processes. Thus, AMPK has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo. The lack of LKB1 or AMPK activation rendered cancer cells more sensitive to cell death induced by glucose deprivation. However, the mechanism by which the failure to activate AMPK accelerates cancer cell death during energy stress remains to be explained. Our results demonstrate that the effect of AMPK does not involve p53 or mTORC1, which were implicated in the LKB1–AMPK-mediated regulation of cell survival during glucose deprivation. In both control A549 cells and dominant-negative p53-expressing A549 cells, LKB1 reconstitution restored AMPK activation and inhibited glucose-starvation-induced cell death to a similar extent. mTORC1 inhibition with rapamycin also did not affect the sensitivity of A549 cells to glucose deprivation. Replacement of glucose with the non-metabolizable glucose analogues 2-deoxyglucose (2DG) or 5-thioglucose (5TG) revealed that only 2DG protected LKB1-deficient cancer cells from glucose-starvation-induced cell death. Unlike 5TG, 2DG strongly induced the activation of AMPK, even in LKB1-deficient cells. This activation occurred by means of a poorly understood mechanism that is dependent on hexokinase. The results suggest that, by activating AMPK, 2DG inhibits cell death during glucose deprivation. However, LKB1 reconstitution in A549 cells induced higher AMPK activation than 2DG, yet 2DG protected the cells slightly better than LKB1, implying that 2DG could protect cells by both AMPK-dependent and AMPK-independent mechanisms. Glucose limitations could induce oxidative stress by decreasing NADPH generation in the pentose phosphate pathway. NADPH is required for the regeneration of reduced glutathione (GSH), which is usedAMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress AMPK is a key regulator of metabolic adaptation, particularly under energy stress conditions that lower intracellular ATP levels. This study shows that AMPK activation during energy stress prolongs cell survival by regulating redox balance. Under these conditions, NADPH generation via the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. AMPK inhibits the acetyl-CoA carboxylases ACC1 and ACC2, which reduces NADPH consumption in fatty-acid synthesis and increases NADPH generation through fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas activation of ACC1 or ACC2 attenuates these processes. Thus, AMPK has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo. The lack of LKB1 or AMPK activation rendered cancer cells more sensitive to cell death induced by glucose deprivation. However, the mechanism by which the failure to activate AMPK accelerates cancer cell death during energy stress remains to be explained. Our results demonstrate that the effect of AMPK does not involve p53 or mTORC1, which were implicated in the LKB1–AMPK-mediated regulation of cell survival during glucose deprivation. In both control A549 cells and dominant-negative p53-expressing A549 cells, LKB1 reconstitution restored AMPK activation and inhibited glucose-starvation-induced cell death to a similar extent. mTORC1 inhibition with rapamycin also did not affect the sensitivity of A549 cells to glucose deprivation. Replacement of glucose with the non-metabolizable glucose analogues 2-deoxyglucose (2DG) or 5-thioglucose (5TG) revealed that only 2DG protected LKB1-deficient cancer cells from glucose-starvation-induced cell death. Unlike 5TG, 2DG strongly induced the activation of AMPK, even in LKB1-deficient cells. This activation occurred by means of a poorly understood mechanism that is dependent on hexokinase. The results suggest that, by activating AMPK, 2DG inhibits cell death during glucose deprivation. However, LKB1 reconstitution in A549 cells induced higher AMPK activation than 2DG, yet 2DG protected the cells slightly better than LKB1, implying that 2DG could protect cells by both AMPK-dependent and AMPK-independent mechanisms. Glucose limitations could induce oxidative stress by decreasing NADPH generation in the pentose phosphate pathway. NADPH is required for the regeneration of reduced glutathione (GSH), which is used