The recombinant human Smad7 adenoviral vector was constructed by direct DNA cloning protocol and then transfected into 293 cells for virus packaging. After amplification and purification, the recombinant adenovirus was used to infect the keloid fibroblasts. The Smad7 mRNA transcription of the infected cells was detected by RT-PCR. The recombinant Adeno-Smad7 was correctly constructed and confirmed by both restriction analysis and PCR analysis. RT-PCR showed the over expression of adenovirus mediated Smad7 mRNA in keloid cells. These results demonstrated that the recombinant Smad7 adenoviral vector can be expressed in cultured cells in vitro, and it may provide a new therapeutic strategy for keloid gene therapy.