Aim To investigate the antagonistic effect of endostatin (ENDO)-vascular endothelial growth inhibitor (VEGI) recombinant adenoviruses to the vascular endothelial cell injury caused by homocysteine (Hcy). Methods The Ad-hENDO-VEGI151 from the fusion protein of ENDO and VEGI was used to transfect the human ECV304 caused by Hcy. The expression of fusion protein was detected by Western blot, the leakage of lactate dehydrogenase (LDH) was detected by automatic biochemical analyzer, the cell viability was detected by trypan-blue staining method, and the expression of tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were detected by ELISA. Results The transgenic efficiency of recombinant adenovirus was relatively high. A fusion protein with molecular weight of 41 kDa was expressed in the Ad-hENDO-VEGI151 recombinant adenovirus of ECV304 cells. Hcy (0.1~1.0 mmol/L) increased the leakage of LDH and decreased the cell viability in concentration-dependent manner. The recombinant adenovirus effectively inhibited the injury of Hcy on human vascular endothelial cells (P＜0.05). Conclusion The ENDO-VEGI recombinant adenoviruses had antagonistic effect on vascular endothelial cell injury caused by Hcy, which might provide an idea for future prospecting.