Retinoic acid receptors (RARs) belong to the nuclear receptor superfamily. The mechanism of ligand-dependent activation of RARs is well known. The effect of protein phosphorylation on the activity of RARs has also been demonstrated. However, it is unclear whether other types of modifications exist and if so whether they can affect the activity of RARs. In a mass spectrometric analysis of mouse RARα expressed in insect cells, we identified a trimethylation site on Lys347 in the ligand binding domain. The modification site was verified in mammalian cells, and site-directed mutagenesis studies revealed the functionality of LyS347 methylation in vivo. Constitutive negative mutants, mimicking hypomethylated RARα, were prepared by replacing methylated LyS347 with either alanine or glutamine. A constitutive positive mutant partially mimicking the hypermethylated RARα was generated by replacing the methylated lysine residue with phenylalanine, a bulky hydrophobic amino acid, to introduce a site-specific hydrophobicity similar to that contributed by lysine methylation. Studies of these mutants revealed that trimethylation of Lys347 of RARα facilitated its interactions with cofactors p3OO/ CREB-binding protein-associated factor and receptor-interacting protein 140 as well as its heterodimeric partner retinoid X receptor, suggesting that site-specific hydrophobicity at LyS347 enhanced molecular interaction of RARα with its modulators. This study uncovers the first example of lysine trimethylation on a mammalian non-histone protein that has an important biological consequence. Our finding also provides the evidence for lysine methylation for the family of nuclear receptors for the first time.