ABSTRACT. Osteosarcoma (OS) causes millions of death worldwide and, since there is no effective therapy, it is necessary to identify the molecular mechanism of OS, which can direct the development of new therapies. This study investigated the role of bone morphogenetic protein 9 (BMP9), a member of the transforming growth factor (TGF)-β family, in OS development. This study first examined BMP9 expression in tissue from OS patients and normal subjects. The OS cell line (MG63) and tumor cells from OS patients were then transfected with BMP9 and cell proliferation and apoptosis were assessed. Western blot and reverse transcription-polymerase chain reaction were used to study the expression of cancer-related genes [B cell lymphoma (Bcl)-2, cleaved Caspase-3, Caspase-9, and poly ADP-ribose polymerase]. To confirm the in vivo impact of BMP9, mice were transplanted with OS tumor cells and then treated with BMP9 carried in attenuated Salmonella enterica serovar Typhimurium. Our study found that the OS tumor tissue had a lower expression of BMP9 compared to normal tissue. Transfection of BMP9 in OS and MG63 cells inhibited cell growth and promoted apoptosis. In vitro studies showed a decrease in Bcl-2 gene expression and an increase in Cyto-c, Caspase-3, and Caspase-9 expression. In vivo studies indicated that consistent treatment with BMP9 in OS mice results in inhibition of tumor growth. This study shows that BMP9 inhibition is associated with OS development and that enhanced expression of BMP9 may be a potential treatment method for OS.