To investigate the effects and mechanisms of berberine (BBR) on the migration, invasion, proliferation and apoptosis of osteosarcoma cells in vitro.Proliferation of MG-63 and U2OS cells was measured by the CCK-8 assay. Cells migration was examined by wound-healing assay. The invasion and metastasis of cells were evaluated by transwell invasion assay. Cells apoptosis was determined by the flow cytometry. Caspase-3 activity in MG-63 and U2OS cells was measured, and Western blot was used to measure the levels of Bax, Bcl-2, MMP-2 and MMP-9 in cells. In addition, the osteosarcoma graft tumor model of mice was established. The tumorigenesis of MG-63 cells in nude mice was compared among three groups. Immunohistochemistry assay was used to measure the levels of MMP-2, MMP-9 and NM-23 in tumor tissue.It was showed that BBR inhibited the proliferation of MG-63 and U2OS cells in vitro in time- and concentration-dependent manners. Moreover, BBR reduced the cells migration and invasion, also down-regulated the expressions of MMP-2 and MMP-9. BBR also inhibited the cells apoptosis by down-regulating the expression of Bcl-2 and up-regulating the expression of Bax. In nude mice, BBR obviously inhibited the tumorigenesis of MG-63 cells. Compared with the negative group, BBR decreased the levels of MMP-2 and MMP-9 and increased the level of NM-23. The molecular mechanism was associated with activation of the MAPK/JNK signal transduction pathway.BBR significantly regulates the biological behaviors of osteosarcoma cells and inhibits the growth of osteosarcoma. The molecular mechanism may be associated with the modulation of MMP/NM-23 and MAPK/JNK signals. BBR may be a potential drug for the treatment of osteosarcoma.AJTR Copyright © 2023.