Cancer cells in culture rely on glutamine as an anaplerotic substrate to replenish tricarboxylic acid (TCA) cycle intermediates that have been consumed. but it is uncertain whether cancers in vivo depend on glutamine for anaplerosis. Here, following in vivo infusions of [13C5]-glutamine in mice bearing subcutaneous colon cancer xenografts, we showed substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors. Consistent with our prior observation that colorectal cancers (CRCs) with oncogenic mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic (PIK3CA) subunit are more dependent on glutamine than CRCs with wild type PIK3CA, labeling from glutamine to most TCA cycle intermediates was higher in PIK3CA-mutant subcutaneous xenograft tumors than in wild type PIK3CA tumors. Moreover, using orthotopic mouse colon tumors estalished from human CRC cells or patient-derived xenografts, we demonstrated substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors and tumors utilize anaplerotic glutamine to a greater extent than adjacent normal colon tissues. Similar results were seen in spontaneous colon tumors arising in genetically engineered mice. Our studies provide compelling evidence CRCs utilizes glutamine to replenish the TCA cycle in vivo, suggesting that targeting glutamine metabolism could be a therapeutic approach for CRCs, especially for PIK3CA-mutant CRCs.