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Bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2, plays a key role in promoting osteosarcoma growth. However, the underlying mechanism remains to be determined. The aim of the present study was to determine the mechanism of BMP9 promoting the growth of osteosarcoma mediated by the Notch signaling pathway. Osteosarcoma cell lines, 143B and MG63, were used for the in vitro experiments. Cell proliferation, cell migration and cell cycle transformation were monitored under various settings. The control and experimental groups used in the present study were BMP9 adenovirus (AdBMP9), a recombinant adenovirus expressing the dominant-negative mutant of Notch1 (AdR-dnNotch1), AdBMP9+AdR-dnNotch1 and AdBMP9+compound E (blocker of the Notch signaling pathway). The results showed that Notch ligands DLL1, JAG1 and JAG2, as well as Notch receptors Notch1, Notch2 and Notch3 were markedly expressed in the two cell lines. Cell proliferation and migration ability increased in the AdBMP9 group and were higher than that in the AdBMP9+AdR-dn Notch1 and AdBMP9+compound E group. Cell proliferation and migration in the AdR-dnNothc1 group was lower than that in the AdBMP9 group, although the differences were not statistically significant (P>0.05). The cell cycle ratio in the S/G2 phase increased significantly in the AdBMP9 group and was higher than that in the AdBMP9+AdR-dnNotch1 and AdBMP9+compound E groups. By contrast, the ratio of the cell cycle in S/G2 phase in the AdR-dnNotch1 group was lower than that in the AdBMP9 group. The differences were not statistically significant (P>0.05). In conclusion, the results showed that the Notch signaling pathway plays an important role in mediating the growth of osteosarcoma promoted by BMP9. [ABSTRACT FROM AUTHOR]