Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.