The objective of this study was to investigate the effect of homoharringtonine on proliferation and apoptosis of osteosarcoma cells through long non-coding RNA (lncRNA) LINC00857/microRNA (miR) -340. U2OS of osteosarcoma cells was treated with harringtonine at a concentration of 0.25, 0.5, 1, 2, 4 μg/ml. P21 and cysteine-containing aspartic protease 3 (Caspase-3) protein expression in U2OS cells were detected by western blot, thiazole blue (MTT) and plate cloning experiments to determine cell survival and colony formation, flow cytometry to measure apoptosis, and quantitative real-time polymerase chain reaction (qRT-PCR) to assay LINC00857 and miR-340 expression. Bioinformatics and dual luciferase reports analyzed the targeting relationship between LINC00857 and miR-340. Cells were transfected with pcDNA3.1-LINC00857 or anti-miR-340, and treated with homoharringtonine to observe their effects on U2OS protein expression, survival, clone formation and apoptosis. We found that different concentrations of homoharringtonine significantly increased the expression of P21, Caspase-3 protein, apoptotic rate, and miR-340 expression in U2OS cells, and obviously decreased the cell survival rate, the number of colony formation, and the expression of LINC00857 (P<0.05). All were dose-dependent. LINC00857 targets and regulates miR-340. Overexpression of LINC00857 or inhibition of miR-340 evidently reduced the expression levels of P21, Caspase-3 and apoptosis in U2OS cells after homoharringtonine treatment, and remarkably improved cell survival rate and colony formation (P<0.05). It is concluded that homoharringtonine can inhibit the proliferation of osteosarcoma cells and induce apoptosis by regulating LINC00857/miR-340.