Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumor associated microenvironment. However, the role of MSC-derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not yet been thoroughly clarified until now. In this study, we established a co-culture model for human bone marrow derived MSCs with osteosarcoma U2OS and MG63 cells, then extraction of exosomes from induced MSCs and study the role of MSC-derived exosomes in the progression of osteosarcoma cell. It was the aim of this study to address potential cell biological effects between MSCs and osteosarcoma cell. we found that MSC-derived exosomes can significantly promote osteosarcoma cells proliferation and invasion. we also found that miR-21-5p were significantly overexpressed in human bone marrow MSCs and MSC-derived exosomes compared with that of human fetal osteoblastic cell line hFOB1.19 by using quantitative realtime polymerase chain reaction (qRT-PCR). Proliferation and invasion of osteosarcoma cells U2OS and MG63 were significantly enhanced by MSC-derived exosomes that were transfected with miR-21-5p. Bioinformatics analysis and dual‐luciferase reporter gene assays validated the targeted relationship between exosomal miR‐21-5p and PIK3R1. Furthermore, we demonstrated that miR-21-5p-abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells U2OS and MG63. In conclusion, Our findings provide new insight into the interaction between MSCs and osteosarcoma cells in tumor associated microenvironment. Notably, the use of a miR-21-5p inhibitor has an excellent restraining effect on osteosarcoma proliferation and invasion, which provides therapeutic potential for osteosarcoma in future clinical medicine.