The high mobility group nucleosome binding domain 5 (HMGN5) is a member of the high mobility group proteins family and highly expressed in multiple malignancies. Nevertheless, the role of HMGN5 in osteosarcoma remains unclear. This study aimed to investigate the expression and clinical significance of HMGN5 in human osteosarcoma, confirm the oncogenic role of HMGN5 and explore the mechanism by which HMGN5 contributes to invasion and metastasis. HMGN5 expression was detected in osteosarcoma tissues and corresponding adjacent non-cancerous tissues (ANCTs) from 52 patients by immunohistochemical (IHC) assay and the clinicopathologic characteristics of all patients were also recorded. Next, osteosarcoma cells were transfected by HMGN5 RNA interference and cell viability, apoptosis and invasion were detected by cell vitality test, flow cytometry and transwell assay, respectively. As a result, IHC assay showed strong immunized activity of HMGN5 in the nucleus in all osteosarcoma tissues compared with the ANCT (53.5 ± 4.3 % vs. 17.0 ± 3.9 %, P < 0.01). HMGN5 expression level was associated with pathologic staging and TNM staging. Knockdown of HMGN5 induced cell cycle arrest, inhibited invasion and increased sensitivity to doxorubicin-induced cell apoptosis in U2-OS and SaO2 cells. Western blot analysis demonstrated that there were increased expressions of cleaved caspase-3, cleaved PARP and decreased expressions of PCNA, PI3Kp85α, p-AKT, MMP-9, and cyclin B1 in U2-OS and SaO2 cells depleted of HMGN5. HMGN5 plays oncogenic role in osteosarcoma by promoting cell proliferation and invasion, and could be exploited as a target for therapy in osteosarcoma.