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Osteosarcoma (OS) refers to a malignant tumor with potential invasiveness and metastasis; however, the current chemotherapy of OS is lacking. Thus, the alternative drug for treating OS is urgent to explore. Calycosin (CC) is evidenced in our previous study to play the anti-OS benefits for suppressing cancer cell proliferation. Consequently, further investigation of CC-medicated anti-invasive and metastatic effects against OS is needed. In the current study, the clinical samples of OS patients were collected for biological and staining assays, such as enzyme-linked immunosorbent assay and polymerase chain reaction. Meanwhile, the cell line and tumor-bearing nude mice were employed in assessing antimetastatic effects of CC against OS through biochemical tests and immunoassays. As a result, the OS patients exhibited upregulated neoplastic expressions of matrix metalloproteinase 2 (MMP2) and proliferating cell nuclear antigen (PCNA), cellular mRNAs and proteins of inhibitor of nuclear factor kappa-B alpha (IκBα), and epithelial cell transforming sequence 2 (ECT2). In cell-line study, CC-treated human OS cells exhibited induced cell apoptosis, reduced cell proliferation, and cellular MMP2 and PCNA concentration, inhibited cell migration, lowered expressions of IκBα ECT2 mRNAs, and proteins. In tumor-bearing nude mice study, CC-treated mice resulted in the dose-dependent reductions of tumor weights and intracellular MMP2 contents. As shown in further assays, neoplastic expressions of interleukin 6 protein, IκBα, ECT2 mRNAs, and proteins were downregulated dose-dependently in CC-treated tumor-bearing mice. In conclusion, these investigative findings suggest that CC may play the potential anti-invasive benefits against OS through suppressing metastasis-associated IκBα/ECT2 molecular pathway., (© 2019 International Union of Biochemistry and Molecular Biology.)