In this study, the promotion of in vitro chondrogenesis was investigated by using chitosan scaffolds and rat bone marrow-derived mesenchymal stem cells (rBMSCs) which are transfected by BMP6 (bone morphogenetic protein-6) encoding gene. For this purpose, plasmid DNA (pShuttle-rBMP6), the expression vector consisting of the coding sequence of the BMP6 was obtained, and then, it was entrapped in chitosan scaffolds to obtain a gene-activated matrix (GAM). The chitosan scaffolds performed the controlled and sustained release of plasmid DNA, thus they continuously provided the modification of rBMSCs to induce chondrogenic differentiation. In addition, the cells were transfected by lipid-based agent (Lipofectamine) and then, these modified cells were inoculated into the chitosan scaffolds. Furthermore, a group of chitosan scaffolds with nontransfected rBMSCs with recombinant BMP6 free in culture medium was used as control. Comparative results showed that, mitochondrial activities of modified rBMSCs by Lipofectamine and chitosan GAM were significantly higher than those of nontransfected rBMSCs. The observations from scanning electron microscopy analysis confirmed that BMP6 gene-modified rBMSCs differentiated to the chondrogenic phenotype. Highest amount of glycosaminoglycan contents of rBMSCs on GAM concluded that BMP6 gene-activated chitosan scaffold has a potential in the application of cartilage regeneration. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.