Development of a novel angiogenesis inhibitor will be essential for the improvement of therapeutics against cancer. Kallistatin had been recognized as an endogenous angiogenesis inhibitor. Here, we demonstrated kallistatin's strong anti-angiogenesis and anti-metastasis activity stimulated by breast cancer cells (MCF-7) and its mechanism of action in vitro. The anti-angiogenesis effect in vivo was evaluated by chicken chorioallantoic membrane (CAM) neovascularisation. Because of the underlying molecular mechanism of its anti-angiogenesis activity remains poorly understood. In this study, we examined whether the NF-κB signaling pathway was involved in the anti-angiogenesis and anti-metastasis activity of kallistatin. Kallistatin significantly inhibited TNF-α-induced nuclear factor-κB activation in a dose-dependent manner. Addition of kallistatin inhibited TNF-α induced IκBα degradation; phosphorylation of IκBα kinase (IKK), nuclear factor-κB-p65 protein; and nuclear translocation of p65/50. Meanwhile, we investigated the effects of kallistatin on the expression of vascular endothelial growth factor (VEGF) and other angiogenesis-related gene in human umbilical vein endothelial cells (HUVECs). We found that kallistatin decreased the expression of VEGF and some angiogenesis-related genes, which promoted angiogenesis in cancer. Taken together, we suggested that kallistatin would inhibit tumor angiogenesis via inhibition of the NF-κB signaling pathway and finally abrogate NF-κB-dependent gene expression. All the results revealed that kallistatin would have potential as a novel.