Intermittent fasting has been reported to exhibit preventive and delaying effects on senescence and age-related diseases by modulating metabolic pathways. To investigate the potential preventive effects of intermittent fasting on retinal ganglion cell loss caused by age-related retinal degenerative diseases such as glaucoma and diabetic retinopathy, we examined the effect of 24-hour fasting on NMDA-induced retinal ganglion cell death in mice. Mice were subjected to 24-hour fasting followed by intravitreal injection of NMDA (10 nmol). Histological analysis revealed that fasting for 24 hours attenuated the loss of retinal ganglion cells induced by intravitreal administration of NMDA in mice. Additionally, Western blotting demonstrated that phosphorylation of AMPKα (Thr172) and FoxO3a (Ser413), which are activated by starvation and contributes to neuroprotection, was enhanced in the retinas of mice subjected to 24-hour fasting. These findings suggest that fasting alleviates NMDA-induced retinal ganglion cell death in mice and intermittent fasting may serve as a potential preventive strategy for retinal neurodegenerative diseases such as glaucoma and diabetic retinopathy.