Angiogenesis plays an important role in cancer growth, invasion and metastasis. It has been confirmed that metadherin (MTDH) is associated with angiogenesis. However, the detailed mechanism of MTDH on angiogenesis has not yet been reported. In this study, we demonstrate the anti-angiogenic function of MTDH in breast cancer. With RNA interference strategies, we found that knockdown of MTDH inhibits cellular angiogenesis both in vitro and ex vivo. Furthermore, we revealed that ERK1/2 pathway is involved in the anti-angiogenic function of MTDH, and the function can be partially reversed via upregulation of microRNA-21 (miR-21). In conclusion, knockdown of MTDH can inhibit angiogenesis in breast cancer. These results show that MTDH is a viable therapeutic target for anti-angiogenesis in breast cancer.