Mesenchymal stem cells are widely stimulated by transforming growth factor beta-3 (TGFβ3) for chondrocyte differentiation. The objective of our study was to establish a new method for differentiation of human mesenchymal stem cells toward chondrocyte by overexpression of MicroRNA-140 (miR-140), and also this method was compared with method of induction with TGFβ3 in high-cell density culture systems. Mesenchymal stem cells were harvested from bone marrow of human. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were cultured in monolayer culture system and were harvested after days 7, 14, and 21. Real-time PCR was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry technique. BMSCs were transducted with recombinant Lenti virus, and miR-140 was expressed. Immunocytochemical method confirmed the differentiation of BMSC toward chondrocyte with handling cartilage matrix genes. Also real-time PCR showed that after expression of miR-140 in transducted BMSCs significantly increased gene expression of collagen type II and aggrecan and downregulated expression of collagen type I when compared with the mRNA levels measured in nontransducted BMSCs. These results were compatible compared with TGFβ3 induction method as control positive. In this study, we described a new approach and technique that may be applied for differentiation of BMSCs to chondrocyte instead of stimulation with TGFβ3. Our data implies that miR-140 is a potent chondrogenic differentiation inducer for BMSCs, and we have shown increasing chondrogenic differentiation by using miR-140 overexpression.