Objective To construct a recombinant adenovirus vector carrying hSulf-1 gene and to investigate its effect on the proliferation and migration of human umbilical vein endothelial cells. Methods The hSulf-1 protein was inserted into adenovirus genome to generate recombinant adenovirus Ad5-hSulf1, and then the virus was used to infect ECV-304 cell line. The expression of hSulf-1 protein was detected by Western blotting analysis, the cell viability was examined by MTT assay, and the cell migration ability was evaluated by wound-healing assay in ECV-304 cells. Results The recombinant adenovirus Ad5-hSulf1 was successfully constructed. Western blotting analysis showed that over-expression of hSulf1 down-regulated the phosphorylation levels of Akt and ERK in ECV-304 cells. MTT assay showed that over-expression of hSulf1 inhibited the proliferation of ECV-304 cells, with the cell survival rate decreased to (68.49±0.05)% at MOI of 50 pfu/ml and (67.78±0.06)% at MOI of 100 pfu/ml(P<0.05). Wound-healing assay showed that over-expression of hSulf1 significantly inhibited the migration of ECV-304 cells compared with the control group (P<0.01). Conclusion The recombinant adenovirus Ad5-hSulf1-mediated hSulf-1 over-expression can markedly inhibit the proliferation and migration of ECV-304 cells, laying a foundation for hSulf-1 related gene therapy of tumor and angiogenesis-associated diseases.