Crocetin, an aglycone of crocin, is found both in the saffron crocus (Crocus starus L.) and in gardenia fruit (Gardenia jasminoides Ellis). We evaluated the protective effects of crocetin against the retinal ischemia induced by 5 h unilateral ligation of both the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. The effects of crocetin (20 mg/kg, p.o.) on ischemia/reperfusion-induced retinal damage were examined by histological, electrophysiological, and anti-apoptotic analyses. Data for anti-apoptotic analysis was obtained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Using immunohistochemistry and immunoblotting, the protective mechanism mediating the effects of crocetin was evaluated by examining crocetin's effects on the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG; used as a marker of oxidative stress) and on phosphorylations of mitogen-activated protein kinases [MAPK; viz. extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK) and p38], and the redox-sensitive transcription factors nuclear factor-kappa B (NF-κB) and c-Jun. The histological analysis revealed that ischemia/reperfusion (I/R) decreased the cell number in the ganglion cell layer (GCL) and the thickness of inner nuclear layer (INL), and that crocetin inhibited GCL and INL. ERG measurements revealed that crocetin prevented the I/R-induced reductions in a- and b-wave amplitudes seen at 5 days after I/R. In addition, crocetin decreased the numbers of TUNEL-positive cells and 8-OHdG-positive cells, and the phosphorylation levels of p38, JNK, NF-κB, and c-Jun present in the retina after I/R. These findings indicate that crocetin prevented ischemia-induced retinal damage through its inhibition of oxidative stress.