Background: To assess intravitreal injection dose and safety of recombinant adeno-associated virus-mediated gene delivery of human NADH dehydrogenase subunit 4 (ND4) in rabbit eyes. Methods: An open reading frame for human ND4 or adeno-associated virus-green fluorescent protein were fused to the mitochondrial targeting sequence and packed into separate adeno-associated virus capsids. Rabbits of three treatment groups were administered 0.1 mL adeno-associated virus-ND4, 0.1 mL adeno-associated virus-green fluorescent protein or 0.1 mL vehicle via intravitreal injection, respectively. The safety of recombinant adenoassociated virus-mediated gene delivery of human ND4 in rabbit eyes was assessed with a slit-lamp microscope and direct ophthalmoscope, measurements of intraocular pressure and flash visual evoked potential, and optical coherence tomography. The mRNA and protein expressions of human ND4 in the retina of rabbits were determined with real-time polymer chain reaction (PCR), immunofluorescence and Western blot. Results: No complications occurred in any of the three treatment groups after the intravitreal injection. At 1-month post-injection, no significant difference in the mean thickness of retinal nerve fibre layer was found among the three groups. Results of the visual evoked potential test showed that there was no difference in the latency of the visual P100 wave among the three groups. Real-time PCR, immunofluorescence and Western blot analyses verified the expressions of ND4 and green fluorescent protein in the retinal nerve fibre layer. Conclusions: Intravitreal injection of adeno-associated virus-ND4 expression vectors was effectively and safely performed in our study. The data on the dose and method of intravitreal injection from our study will provide a valuable reference for clinical intravitreal injection of adeno-associated virus-ND4 for the treatment of Leber's hereditary optic neuropathy.