PURPOSE. Allopregnanolone is a neurosteroid and powerful modulator of neuronal excitability. The neuroprotective effects of allopregnanolone involve potentiation of y-aminobutyric acid (GABA) inhibitory responses. Although glutamate excitotoxicity contributes to ganglion cell death in glaucoma, the role of GABA in glaucoma remains uncertain. The aim of this study was to determine whether allopregnanolone synthesis is induced by high pressure in the retina and whether allopregnanolone modulates pressure-mediated toxicity. METHODS. Ex vivo rat retinas were exposed to hydrostatic pressure (10, 35, and 75 mm Hg) for 24 hours. Endogenous allopregnanolone production was determined by liquid chromatography and tandem mass spectrometry (LC-MS/MS) and immunochemistry. We also examined the effects of allopregnanolone, finasteride, and dutasteride (inhibitors of 5a-reductase), picrotoxin (a GABAA receptor antagonist), and D-2-amino-5-phosphonovalerate (APV, a roadspectrum N-methyl-D-aspartate receptor [NMDAR] antagonist). RESULTS. Pressure loading at 75 mm Hg significantly increased allopregnanolone levels as measured by LC-MS/MS. Elevated hydrostatic pressure also increased neurosteroid immunofluorescence, especially in the ganglion cell layer and inner nuclear layers. Staining was negligible at lower pressures. Enhanced allopregnanolone levels and immunostaining were substantially blocked by finasteride, but more effectively inhibited by dutasteride and APV. Administration of exogenous allopregnanolone suppressed pressure-induced axonal swelling in a concentration-dependent manner, while picrotoxin overcame these neuroprotective effects. CONCLUSIONS. These results indicate that the synthesis of allopregnanolone is enhanced mainly via NMDARs in the pressure-loaded retina, and that allopregnanolone diminishes pressuremediated retinal degeneration via GABAA receptors. Allopregnanolone and other related neurosteroids may serve as potential novel therapeutic targets for the prevention of pressureinduced retinal damage in glaucoma.