AIM: To determine the safe dose of intravitreal clonidine (IVC), a potential drug for neuroprotection and angiogenesis inhibition in rabbits. METHODS: A total of 28 rabbits were divided into four groups. Three groups received IVC with concentrations of 15 (Group A), 25 (Group B), and 50 (Group C) μg/0.1 mL and the control group (Group D) received 0.1 mL balanced salt solution (BSS). To investigate IVC safety, electroretinography (ERG) was performed at baseline, then at 1, 4 and 8wk after injection. After last ERG, all rabbits were euthanized, their eyes were enucleated and subjected to routine histopathological evaluation, immunohistochemistry for glial fibrillary acidic protein (GFAP) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. RESULTS: Based on ERG, histopathology, GFAP and TUNEL assay findings, 15 μg IVC was determined as the safe dose in rabbit eyes. While, the results of routine histopathology and TUNEL assay were unremarkable in all groups, toxic effects attributed to 25 and 50 μg IVC were demonstrated by ERG and GFAP tests. CONCLUSION: Totally 15 μg clonidine is determined as the safe dose for intravitreal injection in rabbits. Contribution of IVC in neuroprotection and inhibition of angiogenesis deserve more studies.