Autologous chondrocyte implantation (ACI) is commonly used for the treatment of cartilage defects. Since the cell number for transplantation is limited, the expand culture of chondrocytes in vitro is needed. However, the phenotype of chondrocytes is easy to lose in monolayer cultured in vitro. Traditional growth factors such as transformation growth factor -β1 (TGF-β1) have been used for promoting the proliferation and maintained the phenotype of chondrocytes, but the high cost and functional heterogeneity limit their clinical application. It is of significant to develop substitutes that can accelerate proliferation and prevent dedifferentiation of chondrocytes for further study. In our present study, the effect of salidroside on proliferation and phenotype maintenance of chondrocytes and cartilage repair was investigated by performing the cell viability, morphology, glycosaminoglycan (GAG) synthesis, cartilage relative genes expression, macroscopic and histological analyzsis. The TGF-β/smad3 signal which may involve in the protective effect of salidroside on chondrocytes was also detected by ELISA and qRT-PCR assays. The results indicated that salidroside could promote chondrocytes proliferation and enhance synthesis of cartilage extracellular matrix (ECM). Expression of collagen type I was significantly down-regulated which suggesting that salidroside could prevent chondrocytes from dedifferentiation. The in vivo experiments for cartilage repair also indicated that in the treatment of salidroside, chondrocytes used for ACI significantly accelerated the hyaline cartilage repair. While in the absence of salidroside, the repaired cartilage is mainly the fibrous cartilage. Additional experiments demonstrated that salidroside promotes the proliferation and maintain the phenotype of chondrocytes by activate the TGF-β/smad3 signal. Salidroside may be a potential agent for ACI to promote the proliferation and maintain the phenotype of chondrocytes expansion in vitro.