Osteosarcoma is the most common primary bone tumor and occurs most frequently in adolescents. Cancer stem cells (CSCs) are resistant to chemotherapy and radiotherapy, and can drive cancer recurrence. in this study, we aimed to investigate the effect of Long noncoding RNAs (lncRNAs) DLX6-AS1 on osteosarcoma sternness and the underlying mechanism involved. DLX6-AS1 enhanced osteosarcoma sternness in vitro and in vivo. Moreover, DLX6-AS1 competitively interacted with miR-129-5p to DLK1, resulting in activation of Wnt signaling and promotion of sternness in osteosarcoma. DLX6-AS1 functionally interplayed with miR-129-5p to form a reciprocal feedback loop to activate Wnt signaling. High DLX6-AS1 expression was observed in osteosarcoma tissues, and predicted a poor prognosis for osteosarcoma patients. Our study suggests that DLX6-AS1, combined with miR-129-5p and DLK1, can be utilized as factors for the clinical diagnosis and prognosis of osteosarcoma, and may be potential targets for the treatment of osteosarcoma. (C) 2018 Elsevier Inc. All rights reserved.