Aim To study the anti angiogenic activity of novobiocin and its mechanism of action. Methods The anti angiogenic activity of novobiocin was determined using chick embryo chorioallantoic membrane(CAM) assay. MTT assay, zymography and related assays were used to observe the effects of drugs on bovine aorta endothelial cells and human pulmonary carcinoma PG cells. Results Novobiocin at the doses of 100 and 200 μg/egg inhibited angiogenesis by 31 6% and 68 7% in CAM, respectively. The combination of novobiocin and vincristine enhanced the anti angiogenic effect. Novobiocin inhibited the proliferation of bovine aortic endothelial cells in a concentration dependent manner. In addition, novobiocin suppressed MMP 2 secretion, migration, and tube formation of endothelial cells. As determined by MTT assay, novobiocin in combination with vincristine displayed synergistic effect on the proliferation of PG cells, Conclusion This study demonstrates that novobiocin is active in suppressing angiogenesis and the anti angiogenic activity may be enhanced by combination with vincristine. The anti angiogenic activity of novobiocin may be related, at least in part, to its inhibition of cell proliferation, cell migration, tube formation and secretion of matrix metalloproteinases.