Current therapies that target vascular endothelial growth factor (VEGF) have become a mainstream therapy for the management of diabetic macular oedema. The treatment involves monthly repeated intravitreal injections of VEGF inhibitors. VEGF is an important growth factor for many retinal cells, including different types of neurons. In this study, we investigated the adverse effect of multiple intravitreal anti-VEGF injections (200 ng/μl/eye anti-mouse VEGF₁₆₄, once every 2 weeks totalling 5–6 injections) to retinal neurons in Ins2^Akita diabetic mice. Funduscopic examination revealed the development of cotton wool spot-like lesions in anti-VEGF treated Ins2^Akita mice after 5 injections. Histological investigation showed focal swellings of retinal nerve fibres with neurofilament disruption. Furthermore, anti-VEGF-treated Ins2^Akita mice exhibited impaired electroretinographic responses, characterized by reduced scotopic a- and b-wave and oscillatory potentials. Immunofluorescent staining revealed impairment of photoreceptors, disruptions of synaptic structures and loss of amacrine and retinal ganglion cells in anti-VEGF treated Ins2^Akita mice. Anti-VEGF-treated WT mice also presented mild amacrine and ganglion cell death, but no overt abnormalities in photoreceptors and synaptic structures. At the vascular level, exacerbated albumin leakage was observed in anti-VEGF injected diabetic mice. Our results suggest that sustained intraocular VEGF neutralization induces retinal neurodegeneration and vascular damage in the diabetic eye.