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Isocitrate dehydrogenase 2 (IDH2) is a mitochondrial NADP-dependent isocitrate dehydrogenase. It is considered to be a novel tumor suppressor in several types of tumors. However, the role and related mechanism of IDH2 in osteosarcoma remain unknown. The expression and significance of IDH2 were investigated by immunohistochemistry in formalin-fixed paraffin sections from 44 osteosarcoma patients. IDH2 was downregulated via lentiviral vector‑mediated RNA interference (RNAi) in the Saos-2 and MG-63 human osteosarcoma cell lines. The effect of IDH2 downregulation on human osteosarcoma was studied in vitro by MTT, flow cytometry and invasion assays. Nuclear factor-κB (NF-κB) and matrix metalloproteinase-9 (MMP-9) assays were also used to study the likely molecular mechanism of IDH2 downregulation on the malignant progression of osteosarcoma cells. The results revealed that the expression of IDH2 was inversely correlated with pathological grade and metastasis in osteosarcoma. IDH2 downregulation promoted a pro-proliferative effect on the Saos-2 and MG-63 osteosarcoma cell lines. IDH2 downregulation accelerated cell cycle progression from S to G2/M phase. The pro-proliferative effect induced by IDH2 downregulation may be ascribed to increased NF-κB activity via IκBα phosphorylation. The invasive activity of osteosarcoma cells was also significantly promoted by IDH2 downregulation and may result from elevated MMP-9 activity. In conclusion, IDH2 downregulation may exacerbate malignant progression via increased NF-κB and MMP-9 activity and may implicate the potential biological importance of IDH2 targeting in osteosarcoma cells. Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.