Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the therapeutic strategies targeting this vulnerability are still inefficient and have unacceptable side effects in clinical trials. Therefore, developing biomarkers to predict therapeutic efficacy would be essential to improve the selective targeting of cancer cells. Here, we found that cell lines that are sensitive to glucose deprivation have high expression of cystine/glutamate antiporter xCT (also known as SLC7A11). We found that cystine uptake and glutamate export through xCT contributed to rapid NADPH depletion under glucose deprivation. This collapse of the redox system oxidized and inactivated AMP-activated protein kinase (AMPK), a major regulator of metabolic adaptation, resulting in a metabolic catastrophe and cell death. Although this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. Taken together, these findings suggest a novel crosstalk between AMPK and xCT that links metabolism and signal transduction, and reveal a metabolic vulnerability to glucose deprivation in cancer cells expressing high levels of xCT.