BCR-ABL1 tyrosine kinase inhibitors (TKIs) are selective therapies for patients with chronic myeloid leukemia (CML) and induce deep molecular response (DMR). However, similar to 60% of patients relapse after the discontinuation of TKIs. Relapse after discontinuation is likely due the inability of TKIs to eradicate CML stem cells (CML-LSCs). In our previous study, 12 out of 22 patients maintained a stable DMR after TKI withdrawal, and we found that fewer patients who were treated with second-generation TKI relapsed compared with those receiving imatinib. Therefore, we hypothesized that second-generation TKIs and imatinib may have different effects on CML-LSCs, which may affect the clinical outcome after TKI discontinuation. To investigate this, we established a TKI discontinuation model in vitro by treating CML CD34(+) cells with imatinib and dasatinib continuously for 72 h and then removing the TKI for 24 h. Colony-forming cell (CFC) assays, apoptosis assessment, and proteomic analysis were then performed. We found that TKI discontinuation resulted in less proliferation and CFC output in dasatinib-treated cells compared with imatinib. However, the dasatinib-treated group exhibited increased apoptosis. In the proteomics analysis, we identified 160 upregulated and 151 downregulated proteins when the two TKI discontinuation groups were compared. Importantly, proteins involved in NAD(+) nucleosidase activity, mitochondrial ATP synthesis coupled proton transport, and oxidative phosphorylation were significantly expressed, which were mainly involved in metabolic processes. In conclusion, we demonstrate that imatinib and dasatinib have clear differential effects on CML-LSCs through the regulation of mitochondria oxidative phosphorylation, which may provide a new target for eliminating CML-LSCs in the context of TKI discontinuation.