Retinal ischemia emerges in many ocular diseases and is a leading cause of neuronal death and dysfunction, resulting in irreversible visual impairment. We previously reported that brain-derived neurotrophic factor (BDNF)-expressing human 293T cells could steadily express BDNF and play a protective role in ARPE-19 cells, a human retinal epithelial cell line. Thus, we hypothesized that exosomes might be essential in the interaction between BDNF-expressing 293T cells and recipient cells. The study investigated whether exosomes derived from BDNF-expressing 293T cells (293T-Exo) can be internalized by ischemic retinal cells and exert neuroprotective roles. The results demonstrated that 293T-Exo significantly attenuated the loss of cell proliferation and cell death in R28 cells in response to oxygen-glucose deprivation treatment. Mechanistic studies revealed that the endocytosis of 293T-Exo by R28 cells displayed dose- and temperature-dependent patterns and may be mediated by the caveolar endocytic pathway via the integrin receptor. In the retinal ischemia rat model, the administration of 293T-Exo into the vitreous humor of ischemic eyes reduced apoptosis in the retina. Furthermore, 293T-Exo was mainly taken up by retinal neurons and retinal ganglion cells. Together, the results demonstrated that 293T-Exo has a neuroprotective effect in retinal ischemia and has therapeutic potential for retinal disorders.