Testicular receptor 4 (TR4) is an orphan member of the nuclear receptor superfamily. Despite the lack of identified ligands, its functional role has been demonstrated both in animals and cell cultures. However, it remains unclear how the biological activity of TR4 is regulated without specific ligands. In this report, we showed that in the absence of specific ligands, the activity of TR4 could be modulated by MAP-kinase (MAPK) mediated phosphorylation of its activation function 1 (AF-1) domain. A mass spectroscopy based proteome analysis of TR4 expressed in insect cells revealed three phosphorylation sites in its AF-1 domain, specifically on Ser19, Ser55, and Ser68. Site-directed mutagenesis studies demonstrated the functionality of phosphorylation on Ser19 and Ser68, but not Ser55. We also demonstrated that MAP-kinase mediated phosphorylation of the AF-1 domain rendered TR4 a repressor, mediated through the preferential recruitment of corepressor, RIP140. Dephosphorylation of its AF-1 made TR4 an activator due to its selective recruitment of coactivator, PCAF. The biological effects were validated by employing the wild type TR4 and its constitutive negative (dephosphorylated) and constitutive positive (phosphorylated) mutants in the studies of regulation of its natural target gene, apo-E. This study uncovered, for the first time, a ligand-independent mechanism underlying the biological activity of TR4, which was mediated by MAPK mediated receptor phosphorylation of AF-1 domain.