Cancer cells rely on glycolysis to maintain high levels of anabolism. However, the metabolism of glucose via glycolysis in cancer cells is frequently incomplete and results in the accumulation of acidic metabolites such as pyruvate and lactate. Thus, the cells have to develop strategies to alleviate the intracellular acidification and maintain the pH stability. We report here that glutamine consumption by cancer cells has an important role in releasing the acidification pressure associated with cancer cell growth. We found that the ammonia produced during glutaminolysis, a dominant glutamine metabolism pathway, is critical to resist the cytoplasmic acidification brought by the incomplete glycolysis. In addition, C-terminal-binding protein (CtBP) was found to have an essential role in promoting glutaminolysis by directly repressing the expression of SIRT4, a repressor of glutaminolysis by enzymatically modifying glutamate dehydrogenase in mitochondria, in cancer cells. The loss of CtBP in cancer cells resulted in the increased apoptosis due to intracellular acidification and the ablation of cancer cell metabolic homeostasis represented by decreased glutamine consumption, oxidative phosphorylation and ATP synthesis. Importantly, the immunohistochemistry staining showed that there was excessive expression of CtBP in tumor samples from breast cancer patients compared with surrounding non-tumor tissues, whereas SIRT4 expression in tumor tissues was abolished compared with the non-tumor tissues, suggesting CtBP-repressed SIRT4 expression contributes to the tumor growth. Therefore, our data suggest that the synergistically metabolism of glucose and glutamine in cancer cells contributes to both pH homeostasis and cell growth. At last, application of CtBP inhibitor induced the acidification and apoptosis of breast cancer cells and inhibited glutaminolysis in engrafted tumors, suggesting that CtBP can be potential therapeutic target of cancer treatment. © 2015 Macmillan Publishers Limited All rights reserved.