Diabetes mellitus is a disease caused by innate or acquired insulin deficiency, resulting in altered glucose metabolism and high blood glucose levels. Chronic hyperglycemia is linked to development of several ocular pathologies affecting the anterior segment, including diabetic corneal neuropathy and keratopathy, neovascular glaucoma, edema, and cataracts leading to significant visual defects. Due to increasing disease prevalence, related medical care costs, and visual impairment resulting from diabetes, a need has arisen to devise alternative systems to study molecular mechanisms involved in disease onset and progression. In our current study, we applied a novel 3D in vitro model of the human cornea comprising of epithelial, stromal, and neuronal components cultured in silk scaffolds to study the pathological effects of hyperglycemia on development of diabetic corneal neuropathy. Specifically, exposure to sustained levels of high glucose, ranging from 35 mM to 45 mM, were applied to determine concentration-dependent effects on nerve morphology, length and density of axons, and expression of metabolic enzymes involved in glucose metabolism. By comparing these metrics to in vivo studies, we have developed a functional 3D in vitro model for diabetic corneal neuropathy as a means to investigate corneal pathophysiology resulting from prolonged exposure to hyperglycemia.