Purpose: Half of the chronic myeloid leukemia (CML) patients with sustained deep molecular response suffer from relapse after discontinuation mainly because tyrosine kinase inhibitors (TKIs) cannot eradicate leukemia stem cells (LSCs). In addition, tumor necrosis factor a (TNF-alpha) is highly detected in CML patients. Our aim was to explore whether TNF-alpha is a potential target for LSC elimination. Materials and methods: We applied a CRISPR/ Cas9 gene editing technique, colonyforming cell assay, subcutaneous tumor models, miRNA-seq and liquid chromatography-mass spectroscopy (LC-MS) on metabonomics to explore the feasibility and mechanism of TNF-alpha as a new therapeutic target for CML. Results: We demonstrated that TNF-alpha knockout remarkably decreased the proliferative, colony-forming and in vivo tumorigenesis capacities of the CML K562 cell line. The apoptosis was increased when TNF-alpha knockout cells were cultured with imatinib. The mechanisms involved in the abovementioned phenomena were that TNF-alpha knockout inhibited the citrate cycle and increased starch, sucrose, amino sugar and nucleotide sugar metabolism. In addition, differentially expressed miRNAs between TNF-alpha knockout and control cells were involved in the cell cycle, CML, P13K-Akt and pathways in cancer. Conclusion: We identified that TNF-alpha may serve as a new target therapy for CML and described the metabolic pathways associated with TNF-alpha in CML cells for the first time.