Adenovirus-mediated overexpression of BMP-9 inhibits human osteosarcoma cell growth and migration through downregulation of the PI3K/AKT pathway. Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of signaling molecules and have previously been shown to be associated with the biological behavior of osteosarcoma. However, to date the effects and molecular mechanisms of BMP-9 on osteosarcoma progression are unknown. We performed real-time PCR and western blot analysis to characterize the endogenous expression of BMP-9 in osteosarcoma cell lines. We used a recombinant adenovirus expressing BMP-9 (adBMP-9) to infect osteosarcoma cell lines with relatively low endogenous BMP-9 expression to determine the functional relevance of BMP-9 overexpression to osteosarcoma cell growth and migration in vitro and in vivo, and further investigated the expression levels of Ki-67, matrix metallopeptidase-9 (MMP-9), phosphoinositide 3-kinase p85α (PI3Kp85α) and phosphorylated AKT (p-AKT). As a result, osteosarcoma cell proliferation and migration were significantly diminished by adBMP-9, indicated by MTT and wound-healing assays, and cell apoptosis was markedly induced, indicated by Hoechst 33342/PI assay and Annexin V-FITC apoptosis detection. When BMP-9 expression was enhanced, the expression of PI3Kp85α, p-AKT, Ki-67 and MMP-9 was downregulated in osteosarcoma cells. In addition, the tumor volumes in MG-63 and HOS subcutaneous nude mouse models treated with adBMP-9 were significantly smaller compared to those of the ad-GFP group. These results suggested that the enhanced expression of BMP-9 in osteosarcoma cells by adBMP-9 exerted inhibitory effects on growth and migration of osteosarcoma cells possibly via blockade of the PI3K/AKT signaling pathway.