Objective To investigate the anti-osteosarcoma effect and mechanism of shikonin ( SK). Methods After various concentrations of SK were applied to human U2OS and MG63 osteosarcoma cells and human osteoblastic cell line HFOB1.19 for 24 h or SK 1 μmol / L for 24, 48 and 72 h, CCK-8 was used to assay cell viability. U2OS cells were treated with SK at 0, 0.01, 0.1 and 1 μmol / L and then colony formation assays were employed to assess cell proliferation. Flow cytometry was used to detect apoptosis. Western blot was performed to determine the expression of apoptosis-related proteins. U2OS cells were divided into control, SK1 μmol / L, PI3K activator insulin-like growth factor-1 ( IGF-1), and SK1 μmol / L+IGF-1 groups. Western blot was used to detect phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT), and expression of caspase-3 (cas3), cleaved cas3, Ki67 and stem cell markers SOX-2, OCT-4 and Nanog. Cell spheroid assay was used to detect the ability of cells to form spheres. U2OS cells were injected subcutaneously into the right abdomen to establish a nude mouse model of tumors and verified in vivo. Results The various concentrations of SK had no obvious toxic effects on HFOB1.19 cells, but had significant toxic effects on U2OS and MG63 cells (P<0.05) in concentration- and time-dependent manners. SK at 0.1 and 1 μmol / L significantly inhibited U2OS cell proliferation and stem cell-like characteristics, induced apoptosis (P<0.05), and inhibited phosphorylation of PI3K and AKT proteins (P<0.05). IGF-1 obviously reversed the inhibitory effect of SK on the PI3K/ AKT signaling pathway and its effect on U2OS cell proliferation, apoptosis, and stem cell-like characteristics (P<0.05). In vivo experiments showed that SK significantly inhibited tumor growth (P< 0.05) and downregulated expression of p-AKT in tumor tissues (P<0.05). Conclusions The inhibitory effect of SK on the growth and stem cell-like characteristics of U2OS osteosarcoma cells may be related to inhibition of PI3K/ AKT signaling pathway activation.