Glucocorticoid eye drops are one of the most widely used medications in ophthalmology. However, little is known about the effects of glucocorticoids on corneal epithelial cells that are directly exposed to topically-administered glucocorticoids. Here we investigated the effects of prednisolone, a synthetic glucocorticoid analogue frequently used in the clinic, on corneal epithelial cells. Results showed that prednisolone decreased survival of corneal epithelial cells by inhibiting proliferation and inducing apoptosis in a dose-dependent manner. The levels of mitochondrial reactive oxygen species (mtROS), cleaved caspase-3, and -9 were increased by prednisolone. The effects of prednisolone on apoptosis and mtROS were blocked 1) by the glucocorticoid receptor (GR) antagonist RU-38486, 2) in cells with GR siRNA knockdown, and 3) by treatment with N-acetylcysteine. Transcript levels of pro-inflammatory cytokines were increased in corneal epithelial cells upon hyperosmolar stress, but repressed by prednisolone. In NOD.B10.H2b mice, topical administration of 1% prednisolone increased apoptotic cells in the corneal epithelium. Together, data indicate that prednisolone induces apoptosis in corneal epithelial cells through GR and the intrinsic pathway involving mtROS, caspase-9, and -3. The pro-apoptotic effects of glucocorticoids along with their anti-inflammatory effects should be considered when glucocorticoid eye drops are used in patients with ocular surface disease.