The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor beta (TGF-beta) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-beta-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of alpha1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-beta-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.