Aim To construct lentiviral Hoxa3 vector, observe its transfection efficiency to human umbilical vein endothelial cells (HUVEC), study its effect on cell migration and angiogenesis, and explore the mechanism of Hoxa3 promoting angiogenesis. Methods Human Hoxa3 gene was obtained from the polymerase chain reaction library by gene synthesis, after being inserted into the bone plasmid, the lentiviral Hoxa3 vector was obtained by transfecting three plasmids into 293T cells, and the titer of lentiviral Hoxa3 vector was determined. Transfection of lentiviral Hoxa3 vector to HUVEC was performed to obtain the maximum transfective efficiency. HUVECs were divided into the control group and lentiviral Hoxa3 transfection group. HUVECs migration and tubule formation experiments were carried out to observe the effects of Hoxa3 on HUVEC migration and tubule formation. Western blot was used to detect the changes of urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-14 (MMP-14) protein expression in HUVECs transfected with lentiviral Hoxa3 vector. Results The lentiviral Hoxa3 vector was successfully constructed, and the titer of the virus was 8×10¹¹ TU/L. The transfection efficiency of 30 MOI lentiviral vector to HUVEC was over 99%. Western blot results showed that Hoxa3 could be effectively expressed in HUVEC after transfection of lentiviral Hoxa3 vector into HUVEC. Compared with the control group, transfection of HUVEC with lentiviral Hoxa3 vector significantly enhanced the migration and tubule formation of HUVEC, and significantly increased the expressions of uPAR and MMP-14 proteins (P＜0.05). Conclusion Successfully constructed lentiviral Hoxa3 vector can promote migration and tubule formation of HUVEC, and its mechanism may be related to its up-regulation of uPAR and MMP-14 proteins in HUVEC.