AIM To construct modified promoter of human endothelial nitric-oxide synthase(eNOS) and investigate transcriptional activity of the eNOS promoter via a hypoxia model of human umbilical vein endothelial cells-12(HUVEC-12) stimulated by Cobaltous chloride in vitro. METHODS The pGL2-eNOS-repSP3 vector was constructed by the SP1-replaced SP3 element with using DNA site-directed mutation of PCR. The cells were transfected with pGL2-eNOS-repSP3 vector and normal pGL2-eNOS-p vector, repsectively. Compared with another modified eNOS promoter(pGL2-eNOS-insSP1) the transcriptional activity of pGL2-eNOS-repSP3 vector was determined using a double luciferase reporter gene system by the stimulation of different concentrations of Cobaltous Chloride. RESULTS The pGL2-eNOS-repSP3 vector was constructed successfully. After the stimulation of Cobaltous Chloride, the transcriptional activities of both pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1 increased as the concentration of Cobaltous Chloride increased. But there was not significantly statistical difference between them. CONCLUSION There is no significant difference on transcriptional activities between the two modified vectors, pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1.