Using genome‐wide CRISPR/Cas9 knockout library screening, this study identifies that pyruvate dehydrogenase α (PDHA), pyruvate dehydrogenase β (PDHB), and pyruvate carboxylase (PC) in pyruvate metabolism are crucial to the adaptation of glutamine depletion in HCC cells. The gene discussed is PDHB; the disease is hepatocellular carcinoma.