Co-conjugating chondroitin-6-sulfate (CSC) and dermatan sulfate (DS) to chitosan scaffolds improves chondrocyte differentiation and extracellular matrix (ECM) production. To further elucidate the cellular responses to CSC/DS conjugation, gene expression profiles for the rat chondrocytes cultured on the CSC/DS/chitosan and chitosan-only scaffolds were compared by reverse-transcription PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR). Our data unraveled that the CSC/DS/chitosan scaffold resulted in low-level expression of collagen I, IIA and X and potentiated the aggrecan, collagen II (including collagen IIB) and TIMP3 expression, but downregulated the decorin expression. Therefore CSC/DS/chitosan scaffold maintained the chondrocyte differentiation while minimized de-differentiation and hypertrophy. Furthermore, CSC/DS conjugation affected the expression of 11 genes implicated in 9 signaling pathways (as unveiled by cDNA microarray) and upregulated the expression of TGF-beta1, Sox9, BMP2, PTHrP and Ihh (as confirmed by qRT-PCR). These data suggested that the CSC/DS/chitosan scaffold potentiated the TGF-beta and Hedgehog pathways, which activated the expression of PTHrP and its downstream Sox9. The signals were transduced to elevate the expression of aggrecan, collagen II and TIMP3, and contributed to the well-differentiated chondrocyte phenotype. Altogether, this study for the first time elucidated the roles of GAGs-conjugated biomaterials in matrix production and breakdown, cellular differentiation and signal transduction at the molecular levels.