Although tumor therapy has been improved in the past decades, the survival outcomes for osteosarcoma remain unsatisfactory, and one of the primary reasons for the failure of current treatment is that patients with late-stage cancer often develop resistance to anticancer drugs. High-mobility group nucleosome-binding domain 5 (HMGN5) is a newly identified gene associated with cancer and autophagy, which could inhibit apoptosis induced by anticancer agents. However, it is still unclear whether HMGN5 regulated autophagy in osteosarcoma, and the mechanism and significance of HMGN5-mediated autophagy in tumor therapy is never investigated. In this study, we first detected HMGN5 in vivo and in vitro. HMGN5 was highly expressed in osteosarcoma tumor, especially in posttreatment tumor. Next, we employed adenovirus-mediated overexpression of HMGN5 in U-2OS and MG63 to investigate the role of HMGN5 in osteosarcoma cell lines. Adenovirus-mediated overexpression of HMGN5 could efficiently upregulate the expression level of HMGN5 in osteosarcoma cell lines at both messenger RNA (mRNA) and protein levels. Anticancer agents namely doxorubicin, cisplatin, and methotrexate each induced HMGN5 upregulation in human U-2OS and MG63 osteosarcoma cell lines. In addition, overexpression of HMGN5 reduced the chemosensitivity of osteosarcoma cells in vitro, and the mechanistic investigation revealed that HMGN5 increased drug resistance by upregulating autophagy. Therefore, HMGN5 is a critical factor in the development of chemoresistance through regulating autophagy, and it offers a novel target for improving osteosarcoma therapy.