Glucocorticoid receptor β (GRβ) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRβ functions as a dominant negative inhibitor of GRα at a molecular level， we determined the crystal structure of the ligand binding domain of GRβ complexed with antagonist RU-486. The structure reveals that RU-486 binds in the same ligand binding pocket as in GRα and the unique C-terminal amino acids of GRβ are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRβ do not contribute to RU-486 binding. Intriguingly， the GRβ/RU-486 complex binds corepressor peptide with similar affinity as GRα/RU-486 complex， despite the lack of helix 12. Our biophysical and biochemical analysis reveals that in the presence of RU-486， GRβ is found in a conformation that favors corepressor binding， potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRβ could repress transcription.