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AIM: To construct the TGF-β3 plasmid containing GFP.And investigate the effect of TGF-β3 gene transfer on chondrogenesis of marrow stromal stem cells(MSCs).Methods: TGF-β 3 full length cDNA was cloned from rat embryos with RT-PCR and it was linked with pMD18-T and then subject to amplification,purification and sequence analysis to make sure the correctness of desired gene.TGF-β 3 full length cDNA was cloned again from pMD18-T use the primers with cleavage sites of EcoR Ⅰ and PstⅠ.The plasmid pIRES2-EGFP as well as the cloned fragment with cleavage sites both underwent double enzyme cutting by restriction enzyme EcoRⅠ and PstⅠ,and then the cloned fragment and the carrier were linked with T4 ligase to get the recombination plasmid pIRES2-EGFP-TGF-β 3.PIRES2-EGFP-TGF-β3 was transferred to MSCs using lipsome,and the genetically modified MSCs were cultured with pellet culture in vitro.Then the matrix collagen Ⅱ (COL Ⅱ),collagen X (COL X),Aggrecan (Agc) and proteoglycan was investigated at day 7,14,21 and 28,respectively.Results: pIRES2-EGFP-TGF-β3 was successfully constructed by means of sequence analysis and enzyme cutting identification.The result of Western Blot indicates the successful expression of TGF-β 3 and genetically modified MSCs with TGF-β3 may be able to differentiate into chondrocyte more efficiently in vitro.Conclusion: Gene transfer of pIRES2-EGFP-TGF-β3 can promote the chondrogenesis of MSCs efficiently.