The inhibitory Smad7, a direct target gene for transforming growth factor-β (TGF-β), mediates TGF-β1–induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-β1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-β–activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-β1–induced apoptosis. The expression of Smad7 was required for TGF-β–induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-β1–induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-β1–induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.