Apoptosis is a multi-step mechanism of cell self‑destruction for maintaining cellular homeostatic balance. Accumulating evidence indicates that abnormal apoptosis promotes the evolution and progression of myelodysplastic syndromes (MDS). As a novel cancer-testis antigen, sperm‑associated antigen 6 (SPAG6) has been reported to regulate apoptosis through the tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in the MDS cell line SKM‑1. However, the mechanism of the intrinsic cell death pathway for apoptosis induction by SPAG6 silencing is unclear. In the present study, the in vitro effects of SPAG6 silencing were investigated in SKM‑1 cells through extensive biochemical and molecular approaches. Western blotting and reverse transcription-quantitative polymerase chain reaction were used to detect the expression of SPAG6 and activation of PTEN/PI3K/AKT signal pathway. Additionally, SKM‑1 cells transduced with SPAG6 short hairpin RNA (shRNA) lentivirus were treated with the phosphatidylionositol 3-kinase (PI3K) inhibitor LY294002 or pan caspase inhibitor z‑VAD‑fmk and the apoptosis rates were measured by flow cytometry, and the expressions of associated proteins were examined by western blot analysis. A mouse xenograft model was also used to further evaluate the effects of SPAG6 knockdown on inducing tumor apoptosis in vivo. Lentivirus-mediated knockdown of SPAG6 in SKM‑1 cells increased phosphatase and tensin homolog (PTEN) expression and reduced protein kinase B (AKT) phosphorylation, which in turn resulted in cell apoptosis as evidenced by induced myeloid leukaemia cell differentiation protein Mcl‑1 downregulation, cytochrome c release and increased caspase‑9 expression. Consistently, the PI3K inhibitor LY294002 synergistically enhanced apoptosis of SKM‑1 cells when co-administered with SPAG6 shRNA lentivirus. Furthermore, treatment with the pan caspase inhibitor z‑VAD‑fmk failed to prevent PTEN activation upon SPAG6 knockdown, suggesting that SPAG6-regulated PTEN expression was caspase activation-independent. In addition, SPAG6 knockdown was associated with DNMT1 downregulation, implying that SPAG6 may indirectly control PTEN expression via DNA methylation. Furthermore, tumor tissues from nonobese diabetic/severe combined immunodeficient mice inoculated with SPAG6-shRNA lentivirus pre-infected SKM‑1 cells exhibited significantly elevated apoptosis in the extrinsic and intrinsic pathways. These results demonstrate that SPAG6 silencing induces PTEN expression to regulate apoptosis though the PI3K/AKT pathway, indicating that SPAG6 may be a potential therapeutic target for MDS.