Transcriptional regulation by the androgen receptor (AR) is critical for male sexual development and prostate cancer. In this study, we used an expression cloning strategy to identify molecules that regulate AR-driven transcription. Screening of a human cDNA library resulted in isolation of caspase-8 (Casp8), an initiator caspase that mediates death-receptor-induced apoptosis. Casp8 repressed AR-dependent gene expression independently of its apoptotic protease activity by disrupting AR amino-terminal and carboxy-terminal (N/C) interaction and inhibiting androgen-induced AR nuclear localization. Protein-protein interaction analysis revealed that three motifs in Casp8 specifically interacted with the motifs that are known to be involved in AR N/C interaction. Substitutions of the amino-acid residues critical for AR-Casp8 interactions abolished the Casp8-mediated inhibition of AR transactivation. In addition, knockdown of Casp8 by RNA interference specifically affected the androgen-dependent expression of AR-targeting genes in LNCaP cells. These results indicate that Casp8 has a novel function beyond its known role in the mediation of apoptosis.