Abstract: Background and Objective Previous research has indicated that the overexpression of human tissue kallikrein-1（hTK1）gene played a part in inhibiting vascular smooth muscle cells（VSMC）proliferation and improving vascular restenosis. However,we don’t know whether the inhibitor of matrix metalloproteinase-1（TIMP1）has the same effect on VSMC. In this study,we established a recombinant adenovirus vector which contained both hTK1 and TIMP1gene and observed its expression and effect in rat VSMC. Methods The plasmid pDC316-CMVhTIMP1-EGFP carried promoter CMV and hTIMP1 cDNA full length was digested with endonuclease BglⅡ and SalⅠ,and subcloned to the plasmid pDC316-hTK1 to construct a recombinant plasmid carried hTK1 and hTIMP1double genes.Recombination adenovirus vector was assembled by double gene plasmid and adenovirus backbones plasmid in 293 Acell. The gene transcription and protein expression were examined by real-time PCR and Western blot assays respectively. VSMC proliferation was detected by cell counting and methyl thiazolyl tetrazoliuin（MTT）.Results The construction of plasmid contained both hTK1 and hTIMP1gene was proved to be correct by PCR,double digestion and sequencing analysis. Ad-hTK1-hTIMP1 could be successfully assembled in 293 Acell. When VSMC were infected with Ad-hTK1-hTIMP1,the increase of transcription and protein expression of hTK1 and hTIMP1 were multiplicity of infection（MOI）-dependent（50-150 MOI）,and the transcription of hTK1 and hTIMP1also increased along with time（1-3d）. Compared with single gene vector（Ad-hTK1 or Ad-hTIMP1alone）,Ad-hTK1-hTIMP1 significantly inhibited the VSMC proliferation（P＜0.01）,with a peak inhibition rate of45.7% at 72 h. Conclusion A recombinant adenovirus vector contained double gene of hTK1 and hTIMP1was successfully constructed for the first time. Both the mRNA and protein levels of hTK1 and hTIMP1were high in rat VSMC.Also,hTK1 and hTIMP1gene showed synergistic suppression effect on inhibiting VSMC proliferation,which laid a preliminary tool for polygene therapy in vascular remodeling.