AbstractAdvanced colorectal cancer (CRC) is characterized by a high recurrence and metastasis rate, which is the primary cause of patient mortality. Unfortunately, effective anti‐cancer drugs for CRC are still lacking in clinical practice. We screened FDA‐approved drugs by utilizing targeted organoid sequencing data and found that the antifungal drug itraconazole had a potential therapeutic effect on CRC tumors. However, the effect and mechanism of itraconazole on CRC tumors have not been investigated. A cell line‐derived xenograft model in tumor‐bearing mice was established and single‐cell RNA sequencing was performed on tumor samples from four mice with or without itraconazole treatment. The proportion of cell populations and gene expression profiles was significantly different between the two groups. We found that itraconazole could inhibit tumor growth and glycolysis. We revealed that CEBPB was a new target for itraconazole, and that silencing CEBPB could repress CRC glycolysis and tumor growth by inhibiting ENO1 expression. Clinical analysis showed that CEBPB expression was obviously elevated in CRC patients, and was associated with poor survival. In summary, itraconazole treatment remodeled cell composition and gene expression profiles. Itraconazole inhibited cell glycolysis and tumor growth via the CEBPB–ENO1 axis. In this study, we illustrate a new energy metabolism mechanism for itraconazole on tumor growth in CRC that will provide a theoretical basis for CRC targeting/combination therapy.