Endostatin (EDN), an endogenous angiogenesis inhibitor of 20kDa. was originally isolated from the supernatant of culture murine hemangioendothelioma cell line. Interest in EDN arises from its therapeutic potential its anti-tumor and anti-angiogenesis agents. However, it is difficult to obtain sufficient quantities of native EDN from its natural resources. We report here the construction of a pMIRH-type vector pLRG29 by introducing the 25s rDNA of Hansenua wingei and the Kin R gene into the YIp vector pSML12 and the EDN expression vector pLRG-EDN by cloning the human EDN cDNA into pLRG29, Human EDN was expressed in Hansenula polymorpha (H.p.) A16 (pLRG-EDN) as it secreted soluble protein, The yield of the secreted EDN was 65 mg/L in shake flask. The secreted EDN was purified to it purity of 98%. by the use of SP Sepharose FF ion-exchange chromatography and Sepharose-heparin Hi Trap affinity chromatography. The MTT and chicken embryo chorioallantoic membrane assay demonstrated that the human EDN produced from H. polymorpha inhibited in vitro the proliferation of human umbilical vein endothelial cells and in vivo the neovascularization induced by bFGF. (c) 2004 Elsevier Inc. All rights reserved.