Objective:To investigate the effect of RNA interference-based silencing of HIF-1α gene on the expression of glycolysis associated genes in esophageal squamous carcinoma cells(Eca-109) cultured in either a normoxia or a hypoxia environment. Methods:Eca-109 cells were divided into three groups,namely,untransfected Eca-109 cells,Eca-109 cells stably transfected with HIF-1α siRNA and empty expression vector. After the cells were cultured in a normoxia environment or in a hypoxia environment for 6,12,24,48 h,respectively,the expression of HIF-1α protein was detected by Western blot,and the expression of glycolysis associated genes(Glut-1,LDH-A,HK-Ⅱ) was determined by RT-PCR and Western blot. Spectrophotometry was employed to determine the activities of the glycolysis enzymes(LDH,HK). Results:After Eca-109 cells were cultured in a hypoxia environment,the expression of HIF-1α protein was upregulated,and expression of HIF-1α was hardly detected in HIF-1α silenced cells,and could not be upregulated in a hypoxia environment. The expression of Glut-1,LDH-A,HK-Ⅱ mRNA and proteins was significantly down-regulated in HIF-1α silenced cells,and the LDH and HK activities were obviously decreased in HIF-1α silenced cells. Conclusion:RNA interference targeting the HIF-1α gene is able to efficiently silence the expression of HIF-1α gene in human esophageal squamous carcinoma cells(Eca-109)and thereby inhibit the expression of glycolysis associated genes(Glut-1,LDH-A,HK-Ⅱ)to different extents.