Background: Recruitment of gene modifying bone marrow mesenchymal stem cells (BMSCs) has been considered an alternative to single-cell injection in articular cartilage repair. Purpose: This study aimed to investigate whether the effect of runt-related transcription factor 2(Runx2) overexpression bone marrow mesenchymal stein cells in vivo could improve the quality of repaired tissue of a knee cartilage defect in a rabbit model. Methods: Thirty-two New Zealand rabbits were randomly divided into four groups. The blank group (Con) did not receive anything, the model group (Mo) was administered saline, the simple stem cell group (MSCs) received MSCs injection, and the Runx2 transfection group (R-MSCs) received Runx2 overexpression MSCs injection. After adapting to the environment for a week, a 5 mm diameter cylindrical osteochondral defect was created in the center of the medial femoral condyle. Cell and saline injections were performed in the first and third weeks after surgery. The cartilage repair was evaluated by macroscopically and microscopically at 4 and 8 weeks. Results: Macroscopically, defects were filled and surfaces were smoother in the MSCs groups than in the Mo group at 4" week. Microscopically, the R-MSCs group showed coloration similar to surrounding normal articular cartilage tissue at 8 weeks in masson trichrome staining. The COL-II, SOX9, and Aggrecan mRNA expressions of MSCs were enhanced at 4 weeks compared with R-MSCs, then the expression reduced at 8 weeks, but was still higher than Mo group level (P<0.05). The western blot examination revealed that the COL-Iland SOX9 expression of MSCs was higher than R-MSCs at 4 weeks, then the expression reduced at 8 weeks, but was still higher than the Mo level (P<0.05). The IL-1 beta content in the joint fluid also revealed that cartilage repair with R-MSCs was better than that with MSCs at 8 weeks (P<0.05). Conclusion: The R-MSCs group showed cellular morphology and arrangement similar to surrounding normal articular cartilage tissue, and Runx2 overexpression of MSCs resulted in overall superior cartilage repair as compared with MSCs at 8 weeks.