Acetylation modification regulates the functions of histone and nonhistone proteins, including transcriptional activity, protein interaction, and subcellular localization. Although many nuclear receptors have been shown to be modified by acetylation, whether retinoid X receptors (RXRs) are acetylated and how the acetylation is regulated remains unknown. Here, we provided the first evidence of RXR acetylation by p300 on lysine 145. Acetylation of RXR by p300 facilitated its DNA binding and subsequently increased its transcriptional activity. Furthermore, we discovered that TR3, an orphan receptor, exerted a negative regulation on p300-induced RXR acetylation. TR3 significantly reduced the p300-induced RXR acetylation and transcriptional activity, and such inhibition required the interaction of TR3 with RXR. Binding of TR3 to RXR resulted in the sequestration of RXR from p300. 9-cis retinoic acid, a ligand for RXR, enhanced the association of RXR with TR3, rather than acetylation of RXR by p300. Biological function analysis revealed that the mitogenic activity of RXR stimulated by p300 was acetylation-dependent and could be repressed by TR3. Upon the treatment of 9-cis retinoic acid, RXR was translocated with TR3 from the nucleus to the mitochondria and apoptosis was induced. Taken together, our data demonstrate the distinct regulatory mechanisms of p300 and TR3 on RXR acetylation, and reveal a previously unrecognized role for orphan receptor in the transcriptional control of retinoid receptors.