Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139 amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4 ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.