Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRα1 and TRβ1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly demonstrated that the presence of T3 can specifically increase the number of FN transcriptional initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-β (TGF-β), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-β neutralizing antibody. In an effort to elucidate the signaling pathways involved in the activation of FN by T3, we demonstrated the involvement of the mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-β, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-β signaling pathway but independent of Smad3/4.