Osteosarcoma, the most common primary mesenchymal malignant tumor, usually has bad prognosis inman, with cancer stem-like cells (CSCs) considered to play a critical role in tumorigenesis and drug-resistance.It is known that phosphatidylinositol 3-kinase (PI3K) is involved in regulation of tumor cell fates, such asproliferation, cell cycling, survival and apoptosis. Whether and how PI3K and inhibitors might cooperate inhuman osteosarcoma CSCs is still unknown. We therefore evaluated the effects of LY294002, a PI3K inhibitor,on the cell cycle and apoptosis of osteosarcoma CSCs in vitro. LY294002 prevented phosphorylation of proteinkinase B (PKB/Akt) by inhibition of PI3K phosphorylation activity, thereby inducing G0/G1 cell cycle arrest andapoptosis in osteosarcoma CSCs. Further studies also demonstrated that apoptosis induction by LY294002 isaccompanied by activation of caspase-9, caspase-3 and PARP, which are involved in the mitochondrial apoptosispathway. Therefore, our results indicate PI3K inhibitors may represent a potential strategy for managing humanosteosarcoma via affecting CSCs.