Nuclear receptors regulate gene activation or repression through dynamic interactions with coregulators. The interactions between nuclear receptors and RNA splicing factors link gene transcription initiation with pre-mRNA splicing, providing a coordinated control of the products of gene transcription. Here we report that two RNA splicing factors, PTB-associated splicing factor (PSF) and p54nrb, synergistically form protein complexes with the androgen receptor (AR) in a ligand-independent manner and inhibit its transcriptional activity. PSF does not affect AR protein stability, as in the case of the progesterone receptor, but impedes the interaction of AR with the androgen response element. Both splicing factors interact directly with mSin3A and attract mSin3A to the AR complex in a synergistic manner. The suppression of AR transcriptional activity by PSF and p54nrb is reversed by the inhibition of histone deacetylase activity. These data demonstrated that PSF and p54nrb complex with AR and play a key role in modulating AR-mediated gene transcription.