〔Abstract〕 To explore the effects of different concentrations of oleic acid on human osteosarcoma cell lines 143B and HOS, as well as the impacts of the optimal concentration of oleic acid on cellular lipid droplet synthesis and cell functions. Methods The 143B and HOS cells were treated with varying concentrations of oleic acid (0, 25, 50, 100, and 200 µmol/L) for 48 hours. Following treatment, Oil Red O staining and BODIPY staining were performed to determine the optimal concentration. Subsequently, CCK8 assays and colony formation experiments were conducted to assess the effect of this optimal concentration of oleic acid on the cell proliferation of both cell lines. Transwell migration assays were utilized to evaluate the influence of the optimal concentration on migratory capacity and Transwell invasion assays were utilized to evaluate the invasive ability. Additionally, Western blot analysis was employed to examine the expression levels of epithelial-mesenchymal transition (EMT) markers Epithelial Cadherin (E-cadherin) and Neural Cadherin (N-cadherin) in response to treatment with the optimal concentration of oleic acid. Results Treatment with oleic acid did not induce significant cell death in either 143B or HOS cells; however, an increase in intracellular lipid droplets was observed alongside enhanced proliferation, migration, invasion capabilities as well as EMT transformation potential. Conclusion Oleic acid induces lipid droplet synthesis in osteosarcoma cells which subsequently promotes their proliferation, migration and invasion abilities along with EMT transformation.