Objective ·To explore the effects of the cancer-testis antigen (CTA) family member CT63 on proliferation, differentiation, and tumorigenicity of chronic myeloid leukemia (CML) cells, and uncover the underlying molecular mechanisms. Methods ·The link between CT63 expression and the prognosis of myeloid leukemia patients was analyzed using bioinformatics methods (TCGA database). A K562 cell line with CT63 knockdown was established. The knockdown efficiency of CT63 was confirmed by qRT-PCR and Western blotting. Live-cell imaging and CCK-8 methods were adopted to evaluate the inhibitory effect of CT63 knockdown in CML cells. A subcutaneous tumorigenesis assay in nude mice was conducted to examine the effects of CT63 on tumorigenesis, tumor growth, and differentiation of K562 cells invivo. Phorbol 12-myristate 13-acetate (PMA)-induced monocyte/macrophage differentiation experiment was carried out to investigate the role of CT63 in the differentiation of K562 cells in vitro. Mitochondrial function was assessed to determine the impact of CT63 on CML cells both in vivo and in vitro. Results ·The Kaplan-Meier survival curve indicated that low expression levels of CT63 were correlated with longer survival in patients with myeloid leukemia. Down-regulation of CT63 in K562 cells inhibited proliferation and promoted differentiation. Live-cell imaging and CCK-8 assays displayed that knockdown of CT63 inhibited cell proliferation and extended cell doubling time in K562 cells. In the subcutaneous xenotransplantation model, down-regulation of CT63 inhibited tumor growth in nude mice. K562 cells expressing lower levels of CT63 were more prone to differentiate into monocyte/macrophage both in vivo and in vitro under PMA exposure condition. Knockdown of CT63 suppressed the activity of mitochondrial respiratory chain complex Ⅳ. This led to decreased expression of mitochondrial markers, including cytochrome C oxidase Ⅳ (COX Ⅳ), pyruvate dehydrogenase, succinate dehydrogenase A (SDHA), and voltage-dependent anion channel (VDAC), thus affecting the mitochondrial metabolic activity of K562 cells. Conclusion ·CT63 is related to the prognosis of myeloid leukemia patients. CT63 plays an important role in promoting proliferation and inhibiting differentiation of K562 cells in vivo and in vitro. CT63 serves as a switch to regulate the balance between proliferation and differentiation of CML cells via the modulation of mitochondrial activity.