CYP46A1 is a CNS-specific enzyme, which eliminates cholesterol from the brain and retina by metabolism to 24-hydroxycholesterol, thus contributing to cholesterol homeostasis in both organs. 2-Hydroxypropyl-β-cyclodextrin (HPCD), an FDA approved formulation vehicle, is currently being investigated off-label for treatment of various diseases, retinal including. HPCD was shown to lower retinal cholesterol content in mice but had not yet been evaluated for its therapeutic benefits. Herein, we put Cyp46a1 mice on high fat cholesterol-enriched diet from 1 to 14 months of age (control group), and at 12 months of age, started to treat a group of these animals with HPCD until the age of 14 months. We found that as compared to mature, regular chow-fed Cyp46a1 mice, control group had about 6-fold increase in the retinal total cholesterol content, focal cholesterol and lipid deposition in the photoreceptor-Bruch’s membrane region, and retinal macrophage activation. In addition, aged animals had cholesterol crystals at the photoreceptor-retinal pigment epithelium interface and changes in the Bruch’s membrane ultrastructure. HPCD treatment mitigated all these manifestations of retinal cholesterol dyshomeostasis and altered the abundance of 6 groups of proteins (genetic information transfer, vesicular transport and cytoskeletal organization, endocytosis and lysosomal processing, unfolded protein removal, lipid homeostasis, and Wnt-signaling). Thus, aged Cyp46a1 mice on high fat cholesterol-enriched diet revealed pathological changes secondary to retinal cholesterol overload and supported further studies of HPCD as a potential therapeutic for age-related macular degeneration and diabetic retinopathy associated with retinal cholesterol dyshomeostasis.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.