Transactivation of EGF receptor (EGFR) by angiotensin II (Ang II) plays important roles in the initiation and progression of chronic kidney diseases. Studies suggest that heparin-binding EGF-like factor (HB-EGF) may be a critical mediator in this process, but its role in vivo has not been investigated. In the current study, we found that in response to Ang II-infusion, kidneys from endothelial HB-EGF deletion mice had significantly reduced EGFR activation compared to controls. Meanwhile, it decreased Ang II-infusion related renal injury, as demonstrated by 1) less albuminuria; 2) less glomerulosclerosis; 3) preserved endothelial integrity and decreased podocyte injury, as shown by greater glomerular tuft area and WT1 positive cells, and fewer apoptotic cells measured by cleaved caspase 3 staining; and 4), reduced Ang II-infusion induced renal inflammation as indicated by less infiltration of F4/80 positive macrophage and CD3 positive T lymphocytes and lower levels of proinflammatory cytokines, such as IL-6 and MCP-1 in both serum and kidney. In conclusion, our results suggest that shedding of HB-EGF from endothelium plays an important role in Ang II-induced renal injury by linking Ang II-AT1R with EGFR transactivation. Inhibition of HB-EGF shedding could be a potential therapeutic strategy for chronic kidney disease.