3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms (AKR1C1-AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3alpha-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3alpha-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3alpha-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3alpha-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.