We isolated MED25, which associates with retinoic acid (RA)-bound retinoic acid receptor (RAR) through the C-terminal nuclear hormone receptor (NR) box/LxxLL motif, and increases RAR/RXR-mediated transcription. When tethered to a promoter, MED25 showed intrinsic transcriptional activity in its PTOV domain, which is likely accomplished by direct association with CBP. Reporter assays using dominant negatives of MED25 demonstrated the importance of the N-terminal Mediator-binding and C-terminal domains in CBP and RAR/RXR binding, which affect MED25 activity. Downregulation of MED25 specifically reduced RAR but not thyroid hormone receptor (TR) activity. Stimulation of RAR by MED25 was correlated with enhanced RA cytotoxicity in vivo. Chromatin immunoprecipitation (ChIP) assays revealed the RA-dependent recruitment of MED25 to the RARbeta2 promoter. Recruitment of CBP and TRAP220 was diminished by the overexpression of a MED25 NR box deletion mutant, and by treatment with MED25 siRNA. Time-course ChIP assays indicated that CBP, together with RAR and MED25, is recruited early, whereas TRAP220 is recruited later to the promoter. Our data suggest that MED25, in cooperation with CBP and Mediators through its distinct domains, imposes a selective advantage on RAR/RXR activation.