Abstract: Objective To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1α) and study their expressions in human microvascular endothelial cells (HMVECs). Methods Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1α into gcc (Ala) in pcDNA3.1⁺-HIF-1α to obtain single-site-mutated vector pcDNA3.1⁺-HIF-1α-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1⁺-HIF-1α-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1α mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting. Results DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1⁺-HIF-1α-564Ala and pcDNA3.1⁺-HIF-1α-564Ala- 803Ala were obtained, both of which could produce HIF-1α protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one. Conclusion The recombinant plasmids pcDNA3.1⁺-HIF-1α-564Ala and pcDNA3.1⁺-HIF-1α- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.