Updated project metadata. Palmitoylation is a dynamic process which regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 60 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant involvement of the palmitoylated proteins in lipid transporter activity and receptor-mediated endocytosis, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900 and 816) of Niemann–Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys 799 and 800 caused significant cholesterol accumulation in late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. At the meantime, the mutated proteins were largely located at the endosome/lysosome. Conclusions: PRE cells have abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol transportation, but not the intracellular localization of NPC1.