Intraocular administration of neurotrophic factors has been shown to delay irreversible degeneration of retinal ganglion cells (RGCs). It would be beneficial for the treatment of optic nerve (ON) injury if such neurotrophic factors could be delivered in a less-invasive manner. The dipeptide leucine–isoleucine (Leu–Ile) appears to induce the production of neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), in the brain. We therefore administered Leu–Ile via periocular depot injection in rats and investigated the dipeptide’s ability to induce BDNF and GDNF in the retina and to delay RGC loss in an ON injury model. Poloxamer–alginate hydrogels containing Leu–Ile were injected into the subconjunctival space of intact or ON-injured rats. BDNF and GDNF levels in the retina were determined by an enzyme immunoassay. Survival of RGCs was assessed in retinal flatmounts. Activation of extracellular signal-regulated kinases (ERK) and cAMP response element binding protein (CREB) in the retina was examined by Western blotting. At 2 h after injection of fluorescein isothiocyanate-conjugated Leu–Ile, the fluorescence intensities in the retina were 4.3-fold higher than those in the saline control. Treatment with Leu–Ile significantly increased the retinal levels of BDNF at 6 h and GDNF at 6–72 h after injection. Treatment with Leu–Ile significantly increased RGC survival to 14 days after ON injury and enhanced the activation of ERK at 72 h and CREB at 48 h after injection in the ON-injured retina. These results suggest that periocular delivery of Leu–Ile induces BDNF and GDNF production in the retina, which may eventually enhance RGC survival after ON injury.