Objective:To construct a lentiviral vector containing HHV－6 U94 gene and observe the effect of U94 on proliferation and angiogenesis of vascular endothelial cells. Methods:The U94－6×His fragment was amplified from plasmid pSR2PH－U94 by PCR and cloned into the lentiviral vector (pLenti6.3－MCS－IRES2－EGFP). The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing,and then cotransfected 293T cells with the auxiliary packaging components plasmids to obtain recombinant lentivirus containing U94－6×His gene. Endothelial cells EA.hy926 were infected with the recombinant lentivirus. After the Blasticidin screening and identification by RT－PCR and western blot,the resistant cell clones were selected. The proliferation and tube formation capacity of endothelial cells were examined by CCK－8 and tube formation assay respectively. Results:The U94 gene was successfully cloned to lentiviral vector pLenti－U94－IRES2－EGFP. The recombinant vector was packaged into 293T cells and the titer of the purified recombinant lentivirus was 2.35 × 107 TU/ml. The U94 stably expressing cell line EA.hy926－U94 was obtained after transfection with the recombinant lentivirus and Blasticidin screening. CCK－8 and tubule formation experimental results show that the proliferation and angiogenesis ability of EA.hy926－U94 were deteriorated significantly compared to the negative control cells EA.hy926－NC and normal cells EA.hy926. Conclusion:The pLenti－U94－IRES2－EGFP lentivitral expression vector was constructed and stably expressing human herpersvirus 6 U94 gene EA.hy926－U94 cell line was established successfully. U94 could inhibit endothelial cells proliferation and angiogenesis.