Estrogen receptors α (ERα) and β (ERβ) are ligand-regulated transcription factors that control important biological processes in humans. The endogenous steroid androstenediol possesses estrogenic activity, despite being a precursor of the primary androgen, testosterone. While androstenediol is an agonist of both ERs, it is ∼ 3-fold selective for ERβ. Additionally, it has been reported that androstenediol can repress proinflammatory responses of the central nervous system (CNS) in an ERβ-dependent manner, but the primary estrogen, estradiol (E2), cannot. As no structural characterization of the interaction between ERα or ERβ with androstenediol has been reported, the basis of ERβ selectivity, and whether this is responsible for the anti-inflammatory effects of androstenediol, remains unclear. To address these gaps in knowledge we determined crystal structures of the human ER LBDs (hERα and hERβ) complexed with androstenediol and coactivator-derived peptide. This revealed that androstenediol stabilizes the active conformation of both receptors in the same manner as E2. The binding mode of androstenediol between the hERα and hERβ LBDs is extremely similar, suggesting that subtle differences in the van der Waals interactions mediated by non-conserved residues of the ligand binding pocket confer selectivity toward hERβ. Finally, in both receptors the coactivator-derived peptide occupied the activation function 2 (AF2) surface, as observed for previous agonist-bound hER structures. Therefore, as androstenediol does not induce any distinct structural changes within the hERβ LBD compared to E2, this suggests that the hERβ-dependent anti-inflammatory effects of androstenediol on the CNS are mediated by other factors.