Glucocorticoids have been widely used as a therapeutic drug for sudden sensorineural hearing loss. However, very little is known about the mechanism(s) underlying the protective effect of glucocorticoids against hearing loss. As an approach toward elucidating the mechanism(s), we evaluated the effects of dexamethasone (DEX) treatment on the biosynthesis of GSH in the mouse cochlea in vivo. The systemic administration of DEX led to a significant increase in the total GSH level in the cochlea 2 to 24 h later. This DEX-induced increase in GSH occurred selectively in the spiral ganglion, but not significantly in the lateral wall tissues or in the organ of Corti. Furthermore, RT-PCR analysis revealed that DEX treatment resulted in enhanced expression of gamma-glutamylcysteine synthetase (gamma-GCS), which is the rate-limiting enzyme for de novo GSH synthesis, 1 to 24 h after the treatment. In addition to enhancing GSH biosynthesis, DEX treatment was effective in reducing lipid peroxidation in the cochlea. Taken together, DEX has the ability to facilitate GSH biosynthesis through enhanced expression of gamma-GCS in the cochlear spiral ganglion.