Over-activation of hypoxia inducible factor (HIF)-1α is implicated as a pathogenic factor in chronic kidney diseases (CKD). However, controversy exists regarding the roles of HIF-1α in CKD. Additionally, although hypoxia and HIF-1α activation is observed in various CKD and HIF-1α has been shown to stimulate fibrogenic factors, there is no direct evidence whether HIF-1α is an injurious or protective factor in chronic renal hypoxic injury. The present study determined whether knocking down the HIF-1α gene can attenuate or exaggerate kidney damage using a chronic renal ischemic model. Chronic renal ischemia was induced by unilaterally clamping the left renal artery for 3 weeks in Sprague-Dawley rats. HIF-1α shRNA or control vectors were transfected into the left kidneys. Experimental groups: sham + control vector, clip + control vector, and clip + HIF-1α shRNA. Enalapril was used to normalize blood pressure one week after clamping the renal artery. HIF-1α protein levels were remarkably increased in clipped kidneys, and this increase was blocked by shRNA. Morphological examination showed that HIF-1α shRNA significantly attenuated injury in clipped kidneys: glomerular injury indices were 0.71 ± 0.04, 2.50 ± 0.12, and 1.34 ± 0.11, and the percentage of globally damaged glomeruli 0.02%, 34.3 ± 5.0%, and 6.3 ± 1.6% in sham, clip and clip + shRNA groups, respectively. The protein levels of collagen and α-smooth muscle actin also dramatically increased in clipped kidneys, but this effect was blocked by HIF-1α shRNA. Conclusion: long-term over-activation of HIF-1α is a pathogenic factor in chronic renal injury associated with ischemia/hypoxia.