Guanine nucleotide binding protein-like 3 (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1-null yeasts. To determine the unique function of GNL3L, we identified estrogen-related receptor gamma (ERRgamma) as a GNL3L-specific binding protein. GNL3L and ERRgamma are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRgamma requires the intermediate domain of GNL3L and the AF2-domain of ERRgamma. Gain-of- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRgamma. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRgamma by GNL3L through coactivator competition.