Objective To construct recombinant adenovirus vector containing human transforming growth factor beta 3 (TGF-β3), which was transfected into marrow mesenchymal stem cells(MSCs) and to observe its expression. Methods The cDNA TGF-β3 was intergraded into the shuttle vector of pAdTrack-CMV and recombinated with adenovirus skeleton vector pAdEasy-1 by homologous recombination. Then the product was transfected into package cell HEK293 by lipofedtamine and the recombinant adenovirus expressing the TGF-β3genewas generated. The rabbit’s MSCs were isolated, cultivated, purified, and then transfected with recombinant adenovirus containing the TGF-β3 gene. The green fluorescence protein expression was observed after 10 days, and the TGF-β3 expression was observed in MSCs transfected by recombinated adenovirus with TGF-β3 gene after 4 days. Results PCR showed that TGF-β3 cDNA was inserted into the recombinantadenoviral plasmid. The recombinant virus vectors with TGF-β3 gene were collected by the packaging HEK293 cells. The fusion rate of MSCs was 70%-80% with an intensive adhesion and uninform shape after the cultured 10th day. Fluorescent microscopy and immunocytochemistry demonstrated that TGF-β3 was expressed in MSCs. Conclusion Successful construction of human TGF-β3 recombinant adenovirus and its expression in MSCs provide a basis of research for the gene therapy of wound healing.