The present work was aimed for the preparation of a stable nanostructured lipid carrier (NLC) system for the delivery of N-palmitoylethanolamide (PEA) to the back of the eye. PEA is an interesting natural compound showing anti-inflammatory and neuroprotective activities. The limits of PEA (poor solubility and high instability) justify its nanoencapsulation into drug delivery systems. Two different well-known techniques were compared to formulate NLC: the high shear homogenization technique (HSH) and the method based on a combination of HSH technique and ultrasonication (HSH/US). Nanoparticles were evaluated in relation to mean size, homogeneity, surface charge, and physical stability by Turbiscan technology. Retinal distribution of PEA was carried out in a rat eye after single instillation of PEA-NLC ophthalmic formulation. The novel formulation delivered remarkable levels of PEA to the retina. Lastly, topical administration of PEA-NLC ophthalmic formulation was able to significantly inhibits retinal tumor necrosis factor-α (TNF-α) levels in streptozotocin-induced diabetic rats. The present findings suggest that the novel ophthalmic formulation may be useful for the treatment of retinal diseases such as diabetic retinopathy. Clinical studies are in progress to evaluate this possibility.