The energy reduction-induced death of retinal ganglion cells is associated with many ophthalmic diseases. The present study was designed to investigate the apoptosis pathway of retinal ganglion cells (RGC-5) following acute ATP reduction by using glucose deprivation (GD). RGC-5 cells were cultured in glucose-free or normal DMEM for 3 days. The changes in intracellular ATP and cell viability were monitored by ATP assay and MTT assay. APOPercentage and in situ TUNEL assays were used to determine the cell death pattern. The involvement of oxidative stress was assessed by measuring intracellular ROS generation, the HO-1 expression, the effect of antioxidants, and the ratio of GSSG to total GSH. The activation of p53 and apoptosis markers was evaluated by Western blotting. We found that glucose deprivation caused an acute decline of intracellular ATP level, concomitantly decreasing cell viability. The cell death exhibited typical features indicative of apoptosis, including cell shrinkage, phosphatidylserine externalization and DNA fragmentation. Oxidative stress was involved in the cell death process; an antioxidant significantly protected the cells against glucose deprivation. p53 and apoptosis markers, caspase-3 and PARP-1 were activated after RGC-5 cells were cultured in glucose-free media for 32 h. Z-VAD-fmk, a pan-caspase inhibitor, was sufficient to prevent apoptosis. These results suggest that acute energy reduction induced by glucose deprivation triggers caspase-dependent apoptosis and activates p53. Blocking the critical steps in this cell death pathway may have therapeutic effects, rescuing the retinal ganglion cells from damages associated with acute energy reduction.