Cancer cells exhibit metabolic dependence on mitochondrial glutamine metabolism that provides them with the substrates required for rapid proliferation. Despite the extensive efforts to target this glutamine addiction for therapeutic purposes, the adaptive metabolic responses and the mechanisms whereby cells maintain their unlimited growth remain areas of active investigation. Here we report that mitochondrial glutamate-pyruvate transaminase 2 (GPT2) contributes to cell survival and growth by sustaining the tricarboxylic acid (TCA) cycle anaplerosis after the inhibition of glutaminase (GLS), the first enzyme for mitochondrial glutamine metabolism. We found that elevated reactive oxygen species upon GLS inhibition induce GPT2 expression via activating transcription factor 4. Moreover, inhibition of GPT2 synergized with suppression of GLS activity to induce a pronounced reduction in proliferation and an increase in cell death of cancer cells. Our data uncover GPT2 as an important component of the adaptive metabolic response for glutamine deprivation and indicate that targeting this pathway in combination with GLS inhibition may be an effective therapeutic approach for cancer treatment.