We found that a specific isoform of hepatocyte nuclear factor 4α (HNF-4α), HNF-4α8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-β. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4α8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4α8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4α8 in TGF-β signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-β1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-β1 was attenuated by forced expression of HNF-4α8. HMGA2 has recently emerged as a transcriptional organizer of TGF-β signaling, regulating several key factors involved in epithelial–mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4α as a new target downstream of HMGA2 and assigned a new role to HNF-4α in the TGF-β signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.