Ethylene diamine tetra acetic acid (EDTA) is a chelating component that is able to diminish oxidative reactivity and can be a potential neuroprotective drug in various ocular diseases. For assessing the safety of intravitreal EDTA, 10 rabbits were allocated and divided into 5 groups. Right eyes of the animals received intravitreal EDTA (112.5, 225, 450, 900 and 1800 µg /0.1 ml). Fellow eyes were considered as controls. Clinical examinations and electroretinography (ERG) were performed at the baseline and on day 28. The enucleated eyes were subjected to hematoxylin and eosin (H&E) staining, immunohistochemistry for glial fibrillary acidic protein (GFAP) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Clinical examinations, H&E staining and TUNEL assay were unremarkable. The ERG test did not exhibit any significant alteration compared to the baseline values, except for a significant decrease in just one measurement of the eyes injected with 225 µg EDTA. The mean scores of GFAP immune reactivity in the eyes injected with 112.5 and 225 µg EDTA indicated a non-significant reaction. The scores in higher doses were significant. We suggest intravitreal EDTA with a dose threshold of < 450 µg should be studied for ratification of the safe dose.