Abstract: Objective To detect the effect of costimulatory molecule B7-H3 on the invasion and metastasis of osteosarcoma cells and related mechanism. Methods The expression of B7-H3 in osteosarcoma cells(U-2OS, MG-63 and Saos-2) were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. B7-H3 siRNA or B7-H3 cDNA was transfected in vitro into human osteosarcoma cells using Lipofectamine® 2000. The expressions of B7-H3 mRNA and protein in osteosarcoma cells were analyzed by RT-PCR and Western blot. The invasive capacity of osteosarcoma cells and MMP-2 expression were measured using Transwell invasion assay in vitro. Results All tested cultured osteosarcoma cells constitutively expressed B7-H3 mRNA under normal conditions. After transfection, the level of B7-H3 expression was decreased in B7-H3 siRNA transfected MG-63 cells, compared with control siRNA transfected group(0.131±0.002) vs. (0.504±0.007), P<0.05 and untransfected group(0.131±0.002) vs. (0.613±0.013), P<0.05. Compared with untransfected cells, the invasive ability and the level of matrix metalloproteinase-2(MMP-2) expression of MG-63 cells were decreased significantly by siRNA interference(P<0.05). After transfection of B7-H3 cDNA, the level of B7-H3 expression was increased in transfected Saos-2 cells, compared with vector transfected group(0.701±0.008) vs. (0.512±0.007), P<0.05 and untransfected group(0.701±0.008) vs. (0.403±0.013), P<0.05. Meanwhile, the invasive ability and the level of matrix metalloproteinase-2 (MMP-2) expression of Saos-2 cells were increased significantly after B7-H3 cDNA transfection(P<0.05). Conclusion B7-H3 plays a novel role in the regulation of the invasive and metastatic capacity of osteosarcoma cells by regulating MMP-2 expression.