To investigate the functional effects of resveratrol (RSV) on mesenchymal stem cells (MSCs), we treated MSCs with RSV continuously during ex vivo expansion. MSCs were continuously treated with RSV from passage (P) 0 to P5. A proliferative capacity of RSV-treated MSCs was higher than that of non-treated MSCs and similar with P1-MSCs. Continuous treatment of RSV on MSCs increased the stemness and inhibited the senescence. During chondrogenic differentiation in vitro, RSV-treated MSCs had higher differentiation potential and reduced hypertrophic maturation, which are limitations for hyaline cartilage formation. The histological analysis of micromass demonstrated increased chondrogenic differentiation potential. We further explored the therapeutic effectiveness of this method in a rabbit osteochondral defect model. A rabbit osteochondral defect model was established to investigate the hyaline cartilage regeneration potential of RSV-treated MSCs. Moreover, the cartilage regeneration potential of RSV-treated MSCs was greater than that of untreated MSCs. The expression levels of chondrogenic markers increased and those of hypertrophic markers decreased in RSV-treated MSCs compared with untreated MSCs. Sustained treatment of RSV on MSCs during ex vivo expansion resulted in the maintenance of stemness and enhanced chondrogenic differentiation potential. Consequentially, highly efficient MSCs promoted superior hyaline cartilage regeneration in vivo. This novel treatment method provides a basis for cell-based tissue engineering.