Glutaminolysis has been established as a hallmark of cancer metabolism, which plays a compensatory role for cell survival upon alternative energy metabolism. CPI-613 (devimistat) is an effective pyruvate dehydrogenase (PDH) inhibitor that interrupts mitochondrial TCA cycle to impair energy metabolism in cancer cells. We report here that CPI-613 resulted in alterations in head and neck cancer cell metabolism by rendering the cells with elevated glutaminase1 (GLS1) expression, leading to an increase in glutamine-dependent cell survival. Genetic knockdown of GLS1 augmented the anticancer effect of CPI-613 in head and neck cancer cells due to inhibiting its induced reprogramming of glutaminolysis. Moreover, pharmacological blockade of the glutaminolysis pathway by GLS1 inhibitor CB-839 not only decreased cancer cell viability dependent on glutaminolysis, but also improved the therapeutic effectiveness of CPI-613 in cell culture and animal models. These findings provide novel evidence that alternative pathways of metabolism endowed cancer cells with metabolic stress, and suppressing the related compensatory pathways might achieve synergistic anticancer outcomes.