Background <p>Osteosarcoma (OS) has a high degree of malignancy, is prone to metastasize. This study aims to identify new biomarkers and natural drugs for OS.</p> Methods <p>Open access OS-related datasets in Gene Expression Omnibus database (GSE39262, GSE21257 and GSE16088) were retrieved and analyzed. The genes related to OS progression were screened through differentially expressed gene analysis, weighted gene co-expression network analysis and univariate COX regression analysis. Immune infiltration analysis was performed using the CIBERSORT and ESTIMATE methods. The binding relationship between DExD-box helicase 39&#xa0;A (DDX39A) and the natural drug coumerstrol was verified with molecular docking and cellular thermal shift assay. OS cell lines with <i>DDX39A</i> knockdown and overexpression were constructed respectively, to investigate the functions of DDX39A and coumerstrol. Cell viability, proliferation, apoptosis, cell cycle distribution, migration and invasion abilities were analyzed by CCK-8, EdU, flow cytometry and Transwell assays, respectively.</p> Results <p><i>DDX39A</i> was highly expressed in OS samples and cell lines, associated with the poor prognosis of the patients. In vitro experiments confirmed that knockdown of <i>DDX39A</i> could inhibit the viability, proliferation, migration and invasion ability of OS cells, causing G1/S phase arrest and apoptosis. DDX39A had a good binding ability with coumerstrol. Coumerstrol treatment could inhibit the expression of <i>DDX39A</i> in OS cells and repressed the malignant behaviors of OS cells, while the overexpression of <i>DDX39A</i> reversed this phenomenon.</p> Conclusion <p>DDX39A is a promising biomarker and therapy target for OS, and coumestrol exerts tumor suppressive properties in OS via inhibiting <i>DDX39A</i>.</p>