The long antisense non-coding RNA ANRIL is 3.8 kb in length and serves an important role in the tumorigenesis and progression of a number of malignancies. However, its function in human osteosarcoma remains unknown. The present study investigated the role of ANRIL in human osteosarcoma cell lines and clinical tumor samples. The expression of ANRIL was analyzed in 19 osteosarcoma and paired adjacent non-cancerous tissues using reverse transcription-quantitative polymerase chain reaction. Knockdown of ANRIL expression using lentivirus-mediated small interfering RNA was performed to investigate the role of ANRIL in tumor proliferation and metastasis using MTT, colony formation and transwell assays. The results demonstrated that ANRIL expression was upregulated in osteosarcoma tissues when compared with ANCT samples. In addition, knockdown of ANRIL expression in vitro reduced cell proliferation and invasion. These results indicated that ANRIL is upregulated in osteosarcoma tissues, and may promote the proliferation and metastasis of osteosarcoma cells. Therefore, ANRIL may serve as a novel biomarker and target of novel therapies for the treatment of patients with osteosarcoma.