Recent developments and re-emergence of interferon α (IFN-α) have renewed interest in the therapy for patients with chronic myeloid leukemia (CML). Related molecular mechanism may be the direct effect of IFN-α on CML stem cells. Human mesenchymal stromal cells (hMSCs) are important to protect CML stem cells, and IFN-α was described as a potential inhibitor of hMSCs. However, the exact mechanism remains obscure. PML as a known tumor suppressor locates downstream of the IFN-α pathway, and little is known about the PML gene regulation in hMSCs. The aim of this study was to investigate the effects of IFN-α on hMSCs and defined the role of PML involved in this process. Our results suggested that hMSCs incurred senescence upon IFN-α stimulation, while PML levels were observed significant increased. The recombinant lentiviral vector, which encodes shRNA against PML or full-length PML cDNA, was constructed. By knocking-down and overexpressing PML, we found that PML was indispensable to IFN-α mediated hMSC senescence. The molecular mechanism underlying this process may be an increased co-localization of PML and p53 induced by IFN-α. Our data demonstrated that IFN-α can induce cellular senescence of hMSCs and PML plays a key role in this process. These findings provided novel insight into the effect of IFN-α on hMSCs.