Glutamine supports cancer cell growth, yet cancer cells can survive glutamine depletion. Reid et al. reveal a novel pathway exploited by tumours to adapt to glutamine starvation through the activation of inhibitor of nuclear factor-κB (NF-κB) kinase subunit-β (IKKβ). Independently of inducing NF-κB transcriptional activity, IKKβ directly phosphorylates and inhibits the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), decreasing aerobic glycolysis under low glutamine conditions. The combined inhibition of glutamine metabolism and IKKβ synergistically reduced tumour growth in vivo, suggesting that to target this metabolic adaptation may be beneficial to cancer patients.