The dynamic regulation of integrin-binding peptides is crucial for chondrogenic differentiation. Here, we revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6-6.25) RGD-chimeric protein (CBD-RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of adipose-derived adult stem cells (ADAS). The binding of CBD-RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD-RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrices and decrease of collagen X gene expression during TGF-beta3 induction were only observed for those beads containing 10mg/g CBD-RGD initially, with 20.18+/-0.73% of that released in a week. The contrary was observed for beads with CBD-RGD 20mg/g initially and having higher persistence (only 8.6+/-2.17% released in a week). The 10mg/g CBD-RGD-mediated enhancement was demonstrated via the activation of integrin alpha5 and beta1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD-RGD may account for the enhanced chondrogenesis. It was concluded that the CBD-RGD-alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-beta3 induction in an RGD dose-dependent manner.