Peroxisome proliferator-activated receptor (PPAR)-γ and retinoic acid X receptor (RXR) heterodimer regulates cell growth and differentiation. Zinc finger transcription factor-9 (Zf9), whose phosphorylation promotes target genes, is a transcription factor essential for transactivation of the transforming growth factor (TGF)-β1 gene. This study investigated whether activation of PPARγ-RXR heterodimer inhibits TGFβ1 gene transcription and Zf9 phosphorylation and, if so, what signaling pathway regulates it. Either 15-deoxy-δ(12,14)-prostaglandin J₂ (PGJ₂) or 9-cis-retinoic acid (RA) treatment decreased the TGFβ1 mRNA level in L929 fibroblasts. PGJ₂ + RA, compared with individual treatment alone, synergistically inhibited the TGFβ1 gene expression, which was abrogated by PPARγ antagonists. Likewise, PGJ₂ + RA decreased luciferase expression from the TGFβ1 gene promoter. Promoter deletion analysis of the TGFβ1 gene revealed that pGL3-323 making up to -323-base pair region, but lacking PPAR-responsive elements, responded to PGJ₂ + RA. PGJ₂ + RA treatment inhibited the activity of p70 ribosomal S6 kinase-1 (S6K1), abolishing Zf9 phosphorylation at serine as did rapamycin [a mammalian target of rapamycin (mTOR) inhibitor]. Zf9 dephosphorylation by PGJ₂ + RA was reversed by transfection of cells with the plasmid encoding constitutively active S6K1 (CA-S6K1). Transfection with dominant negative S6K1 inhibited the TGFβ1 gene. TGFβ1 gene repression by PGJ₂ + RA was consistently antagonized by CA-S6K1. Ectopic expression of PPARγ1 and RXRα repressed pGL3-323 transactivation with S6K1 inhibition, which was abrogated by CA-S6K1 transfection. PGJ₂ + RA induced phosphatase and tensin homolog deleted on chromosome 10 (PTEN), whose overexpression repressed the TGFβ1 gene through S6K1 inhibition, decreasing extracellular signal-regulated kinase 1/2-90-kDa ribosomal S6 kinase 1 and Akt-mTOR phosphorylations. Data indicate that activation of PPARγ-RXR heterodimer represses the TGFβ1 gene and induces Zf9 dephosphorylation via PTEN-mediated S6K1 inhibition, providing insight into pharmacological manipulation of the TGFβ1 gene regulation.