The anti-inflammatory molecule annexin A1 (ANXA1) determines the ultimate fate of retinal ganglion cell (RGC) in glaucoma. Cytoplasmic and extracellular ANXA1 facilitate resolution of inflammation. However, the nuclear translocation of ANXA1 induces RGC apoptosis in a murine glaucoma model, and the maintenance of ANXA1 secreted in the extracellular environments remains unclear. In this study, we found that intravitreal injection of the recombinant adenovirus vector (Ad)-ATP-binding cassette transporter A1 (ABCA1; carrying full-length ABCA1) improved RGC survival in the ischemia reperfusion (IR) mice model. Upregulation of ABCA1 maintained ANXA1 cytoplasmic location and reduced ANXA1 nuclear translocation, which is due to the decreased binding of ANXA1 with importin b. Moreover, we found that amino acids 903 to 1,344 of ABCA1 interacted with ANXA1 and decreased its nuclear localization. Importantly, intravitreal injection of adenovirus-associated viral (AAV) vector AAV8-ABCA1 (carrying 903 to 1,344 fragments of ABCA1) maintained ANXA1 cytoplasmic location and improved RGC survival in the IR mice model. Thus, overexpression of ABCA1 protects against RGC apoptosis by partially blocking ANXA1 nuclear translocation. This study puts forth a potential gene treatment strategy to prevent RGC apoptosis in glaucoma.