In this study, we investigated the regulatory mechanism of SPOP expression in PCa especially in terms of CSCs and found that SPOP expression is negatively regulated by SMAD3-mediated TGF-β signaling through the interaction between SMAD3 and its binding elements (SBEs) in the promoter of SPOP. Thus, our study reveals a novel role of TGF-β signaling in regulating SPOP expression and resultant PCa stemness. This evidence concerns the gene SMAD3 and posterior cortical atrophy.