Transforming growth factor-β (TGF-β), a secreted factor present at high levels in bone, inhibits osteoblast differentiation in culture; yet, the mechanism of this inhibition remains unclear. We studied the effects of TGF-β and its effectors, the Smads, on the expression and function of the osteoblast transcription factor CBFA1. TGF-β inhibited the expression of the cbfa1 and osteocalcin genes, whose expression is controlled by CBFA1 in osteoblast-like cell lines. This inhibition was mediated by Smad3, which interacts physically with CBFA1 and represses its transcriptional activity at the CBFA1-binding OSE2 promoter sequence. The repression of CBFA1 function by Smad3 contrasts with previous observations that Smads function as transcription activators. This repression occurred in mesenchymal but not epithelial cells, and depended on the promoter sequence. Smad3-mediated repression of CBFA1 provides a central regulatory mechanism for the inhibition of osteoblast differentiation by TGF-β, since it inhibits both cbfa1 transcription and transcriptional activation of osteoblast differentiation genes by CBFA1. Altering Smad3 signaling influenced osteoblast differentiation in the presence or absence of TGF-β, implicating Smad3/TGF-β-mediated repression in autocrine regulation of osteoblast differentiation.