Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence forming another his-tag structure, was approved by the State Food and Drug Administration of China (SFDA) in 2005 for the treatment of non-small-cell lung cancer. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we examined the anti-invasive activities of endostar in vitro. The results showed that endostar suppressed MDA-MB-435 cell adhesion to the fibronectin-coated substrate in a concentration-dependent manner. It could inhibit the wound healing migration of MDA-MB-435 cells and invasion of MDA-MB-435 cells through reconstituted ECM (matrigel). Zymography revealed that endostar decreased the secretion of MMP-2 and MMP-9. Endostar could also inhibit the expressions of MMP-2 and MMP-9 in MDA-MB-435 cells. Additionally, endostar exerted an inhibitory effect on the phosphorylation of ERK1/2. Collectively, these data provided a molecular basis for the anti-invasive effects of endostar.