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Background: Osteosarcoma (OS) is commonly recognized as a malignant cancer originating from bone-forming mesenchymal stem cells, comprising approximately 20% of sarcomas. Baicalin, a bioactive flavonoid glycoside isolated from Scutellaria baicalensis , has been demonstrated to possess potent anti-inflammatory and neuroprotective properties., Objective: To explore the potential mechanisms through which baicalin exerts anti-osteosarcoma effects and facilitates osteogenesis in vitro ., Methods: Cell Counting Kit-8 (CCK-8), scratch assay, and transwell assay were employed to assess the effects of baicalin at varying concentrations (20, 40, and 80 μM) on U2OS cell proliferation, invasion, and migration, respectively. Western blot and qRT-PCR analyses were conducted to evaluate the influence of baicalin on the osteogenic potential of OS cells by examining osteoblast markers such as osteocalcin ( OCN ), osteopontin ( OPN ), and runt-related transcription factor 2 ( RUNX2 ), as well as the osteoclast marker-receptor activator of nuclear factor kappa B ligand ( RANKL ). Additionally, the impact of baicalin on epithelial-mesenchymal transition (EMT) markers (N-cadherin, E-cadherin, Vimentin) and proteins related to the Nuclear factor κB (NF-κB) signaling pathway (p-p65, p-IκBα, p65, IκBα) in OS cells was evaluated via western blot analysis. The activity and mineralization capacity of Alkaline Phosphatase (ALP) in baicalin-treated cells were examined through ALP staining and Alizarin Red S (ARS) staining., Results: Baicalin exhibited significant suppression of OS cell U2OS invasion ( p < 0.01), migration ( p < 0.01), and proliferation ( p < 0.05) at various concentrations. Additionally, baicalin treatment notably increased the E-cadherin protein level, while decreasing the expression levels of Vimentin and N-cadherin proteins ( p < 0.01), thus promoting EMT. Following baicalin treatment, there was a marked elevation in the protein and mRNA expression levels of RUNX2, OPN, and OCN, while the expression level of RANKL protein was reduced ( p < 0.05), indicating enhanced osteogenic differentiation. The groups treated with baicalin exhibited higher ALP activity and mineralization ability ( p < 0.01). Moreover, baicalin treatment significantly reduced the expression levels of p-IκBα and p-p65 proteins, as well as the ratios of p-IκBα/IκBα and p-p65/p65 ( p < 0.01). These effects of baicalin were concentration-dependent, with higher concentrations yielding stronger effects., Conclusion: In vitro , baicalin demonstrates anti-OS effects and facilitates osteogenic differentiation, potentially by inhibiting NF-κB pathway activity.