Excess iron content can build up in the retina and lead to iron-mediated retinal injury. An important isoflavone C-glucoside, puerarin, has been reported to be involved in retinal protection. In this experiment, we studied the effects and potential mechanisms of puerarin on retinal injury in vivo and in vitro. We found that puerarin reduced serum and retinal iron content, attenuated the pathophysiological changes and retinal iron deposition, and partially prevented the decrease of rhodopsin and retinal pigment epithelium-specific 65 kDa protein expression in retinas of iron-overload mice. Puerarin rescued the abnormal expression of iron-handling proteins in the mouse retina and suppressed the oxidative stress induced by iron overload, as evident from the enhanced activity of superoxide dismutase, catalase, and glutathione peroxidase and decreased content of malondialdehyde. Moreover, puerarin inhibited the phosphorylation of p38 and ERK mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription 3 (STAT3), thereby protecting the retinal cells from apoptosis by suppressing cytochrome c release, caspase activation, and poly (ADP-ribose) polymerase cleavage in vivo. Also, the ability of puerarin to regulate iron-handling proteins, decrease intracellular Fe²⁺, and inhibit cell apoptosis was further confirmed in ARPE-19 cells. The experimental data verify the protective role of puerarin in the treatment of retinal injury caused by iron overload; its possible mechanisms might be associated with regulation of iron-handling proteins, enhancement of the antioxidant capacity, and the inhibition of MAPK and STAT3 activation and the apoptotic pathways under iron overload conditions.