This study reports the three-dimensional structures of the estrogen receptor beta (ERβ) ligand-binding domain (LBD) in complexes with the phyto-oestrogen genistein and the antagonist raloxifene. The overall structure of ERβ-LBD is similar to that of ERα-LBD, with each ligand interacting with a unique set of residues within the hormone-binding cavity and inducing distinct orientations in the AF-2 helix (H12). Raloxifene, a full antagonist, protrudes from the cavity and prevents H12 from aligning over the bound ligand, while genistein, a partial agonist, is buried within the hydrophobic core and binds similarly to the endogenous hormone, 17β-oestradiol. However, in the ERβ-genistein complex, H12 does not adopt the 'agonist' position but lies in a similar orientation to that induced by ER antagonists, suggesting that genistein's partial agonist character in ERβ is due to a sub-optimal alignment of the transactivation helix. These findings provide insights into the ligand-binding properties of the two ER isoforms and their response to certain ligands.This study reports the three-dimensional structures of the estrogen receptor beta (ERβ) ligand-binding domain (LBD) in complexes with the phyto-oestrogen genistein and the antagonist raloxifene. The overall structure of ERβ-LBD is similar to that of ERα-LBD, with each ligand interacting with a unique set of residues within the hormone-binding cavity and inducing distinct orientations in the AF-2 helix (H12). Raloxifene, a full antagonist, protrudes from the cavity and prevents H12 from aligning over the bound ligand, while genistein, a partial agonist, is buried within the hydrophobic core and binds similarly to the endogenous hormone, 17β-oestradiol. However, in the ERβ-genistein complex, H12 does not adopt the 'agonist' position but lies in a similar orientation to that induced by ER antagonists, suggesting that genistein's partial agonist character in ERβ is due to a sub-optimal alignment of the transactivation helix. These findings provide insights into the ligand-binding properties of the two ER isoforms and their response to certain ligands.