Abstract: To evaluate the inhibitory action of endostatin (ES) on human osteosarcoma tumor growth in nude mice and its possible mechanism. Methods: The inhibitory role of ES on endothelial cell migration induced by osteosarcoma cell line 9901 was observed using a transwell model.Thirty Balb/c nu/nu mice implanted with osteosarcoma were randomly divided into the control, ES low dose (0.75 mg/Kg.d) and ES high dose (1.5 mg/Kg.d) groups. ES was injected intratumorally and around the tumor, once a day for 14 days. The tumor volumes were measured and the tumor inhibition rate (TIR) was calculated. The percentages of necrotic tissue, microvessel density, proliferation index (PI) and apoptotic index (AI) were detected by pathohistologic observation and immunohistochemical staining.The mice were sacrificed 3 h after EF5 injection via tail vein, and then the hypoxia of tumor cells and microvessels was observed using laser confocal microscopy. Results: Both the low dose (350 ng/ml) and high dose (700 ng/ml) ES groups showed a marked inhibition of endothelial cell migration induced by osteosarcoma cells, while the high dose showed much stronger inhibition than the low dose (P<0.01).The tumor volume was significantly different among the 3 groups. The TIR in the high dose and the low dose groups were 35.8% and 22.9%, respectively. In the control group, the ES low dose group and the ES high dose group, the percentage of necrotic tissue was 2.40%, 4.68% and 9.52%, the PI was 66.44%, 46.11% and 25.11%, and the AI was 2.69%, 7.58% and 11.53%, respectively (P<0.01). Much less green fluorescence (microvessel) and stronger red fluorescence (hypoxia) were apparent in the tumors treated with ES when observed with laser confocal microscopy. Conclusions: The results show that ES markedly inhibits endothelial cell migration in vitro and significantly restrains the growth and development of osteosarcoma in vivo. The mechanism may be due to anti- angiogenesis properties associated with hypoxia in the tumor and apoptosis of tumor cells.