Transforming growth factor (TGF)-beta has an essential role for the Sry-type high-mobility-group box (Sox)-regulated chondrogenesis. Chondrogenic differentiation is also controlled by chromatin-mediated transcription. We have previously reported that TGF-beta-regulated Smad3 induces chondrogenesis through the activation of Sox9-dependent transcription. However, the cross-talk between TGF-beta signal and Sox9 on chromatin-mediated transcription has not been elucidated. In the present study, we investigated the activity of Smad3, Sox9, and coactivator p300 using an in vitro chromatin assembly model. Luciferase reporter assays revealed that Smad3 stimulated the Sox9-mediated transcription in a TGF-beta-dependent manner. Recombinant Sox9 associated with phosphorylated Smad3/4 and recognized the enhancer region of type II collagen gene. In vitro transcription and S1 nuclease assays showed that Smad3 and p300 cooperatively activated the Sox9-dependent transcription on chromatin template. The combination treatment of phosphorylated Smad3, Sox9, and p300 were necessary for the activation of chromatin-mediated transcription. These findings suggest that TGF-beta signal Smad3 plays a key role for chromatin remodeling to induce chondrogenesis via its association with Sox9.