Abstract: Objective To investigate whether Leech hirudo could inhibit tumor angiogenesis by improving the hypoxia microenvironment and its related anti-angiogenesis mechanisms. Methods Cobalt Chloride was added in the medium to simulate the chemical hypoxia environment.We used MTT method to observe the EA.Hy926 cells proliferation.The mRNA expressions of hypoxia inducible factor 1α(HIF-1α) and vascular endothelial growth factor(VEGF) were examined by real-time PCR,and the protein level of HIF-1α was detected by Western blot.The tube formation assay was employed to detect EA.Hy926 cells angiogenesis in vitro. Results The Leech hirudo medicated-serum inhibited EA.Hy926 cells proliferation obviously in time and dose-dependent manner in the hypoxic environment.The mRNA expressions of hirudo-medicated HIF-1α and VEGF,and the protein level of HIF-1α induced by Leech serum in hypoxia were remarkably inhibited,and significant differences were observed compared with the negative group (P<0.05).The mRNA and protein of HIF-1α in the high medicated group was equal to that of the positive group (P>0.05).However,the expression of VEGF mRNA of the high medicated group decreased significantly compared with the positive group (P<0.05).The tube number declined in both low and high hirudo medicated-serum dose groups,and significant differences were observed compared with negative group (P<0.05),but not in positive group(P>0.05). Conclusion Leech hirudo could inhibit tumor angiogenesis via improving tumor hypoxia microenvironment,which may be attributed to decrease the expression of mRNA and protein level of HIF-1α,and degrade the VEGF,the downstream gene of HIF-1α mRNA expression.