Diabetic retinopathy (DR), a common complication of diabetes, is a leading cause of blindness among working-age individuals worldwide. Traditionally, DR was considered primarily a microvascular complication, categorized as either nonproliferative or proliferative based on neovascularization presence. However, emerging evidence suggested that neurodegeneration plays a crucial role in DR beyond its microvascular aspects. Neurotrophic factors, such as nerve growth factor (NGF), have significant effects on neuronal survival. In the visual system, NGF influences retina and optic nerve development, supporting the survival and recovery of retinal ganglion cells. Additionally, both in vitro and in vivo studies demonstrate NGF’s protective impact on retinal photoreceptors, Müller cells, and vascular pericytes. In this study, NGF treatment was investigated using in vitro and in vivo models of DR. We examined the potential therapeutic effects of NGF and explored the underlying biological and molecular mechanisms. Three retinal cell lines—ARPE-19 (retinal pigmented epithelium), R28 (a neuronal retinal cell model), and rMC-1 (Müller cell line)—were utilized. Furthermore, intraocular administration of NGF was evaluated in Streptozotocin-induced diabetic mice, revealing a significant protective effect of this neurotrophin in DR through electroretinogram analysis, morphological assessment, and protein expression evaluation. This investigation provides valuable insights into the complex interplay between hyperglycemia, retinal cell damage, and the therapeutic potential of rhNGF supplementation in diabetic retinopathy. The promising efficacy of rhNGF in ameliorating DR underscores its potential for further clinical translation and therapeutic development in the field, offering hope for improved outcomes and vision preservation in individuals affected by this debilitating condition.