Compound YSY-01A is a recently synthesized proteasome inhibitor. It has been proved for potent growth-inhibitory effect on tumor cells in previous studies. However, the effect of YSY-01A on tumor angiogenesis remains unclear. Our research aims to reveal the inhibition effect and mechanism of YSY-01A on tumor-induced angiogenesis. Firstly, we combined the Sulforhodamine B (SRB) assay and Transwell co-culture model to observe the inhibition of YSY-01A on human umbilical vein endothelial cells (HUVECs) proliferation induced by tumor cells (HT-29 cells). In succession, high content screening (HCS) assay was used to investigate effect of YSY-01A on NF-κB nuclear translocation in HT-29 cells. Finally, Western blot was used to measure the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in HT-29 cells inhibited by YSY-01A. To further determine mechanism of inhibition, SRB and HCS methods were used to investigate the effect of YSY-01A against HUVECs proliferation and motility, respectively. The results showed that YSY-01A could prohibit HUVECs proliferation induced by HT-29 cells in a concentration-dependent manner. Furthermore, YSY-01A significantly inhibited NF-κB nuclear translocation and reduced the expression of HIF-1α and VEGF in HT-29 cells. Further investigation revealed concentration-dependent suppress of YSY-01A on HUVECs proliferation and motility without obvious cytotoxic effect. In conclusion, through proteasome inhibition, YSY-01A could down-regulate pro-angiogenesis factors expression in tumor cells and exhibit remarkable anti-angiogenesis activity on vascular endothelial cells.