Protein S-acylation (palmitoylation), a reversible post-translational modification, is critically involved in regulating protein subcellular localization, activity, stability, and multimeric complex assembly. However, proteome-scale characterization of S-acylation has lagged far behind that of phosphorylation, and global analysis of the localization of S-acylated proteins within different membrane domains has not been reported. Here we describe a novel proteomic approach, designated Palmitoyl Protein Identification and Site Characterization (PalmPISC), for proteome-scale enrichment and characterization of S-acylated proteins extracted from lipid raft-enriched and non-raft membranes. In combination with label-free spectral counting quantitation, PalmPISC led to the identification of 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate S-acylation sites. Palmitoyl acyltransferases DHHC5, DHHC6, and DHHC8 appear to be S-acylated on three cysteine residues within a novel CCX7~13CS/T motif downstream of a conserved Asp-His-His-Cys cysteine-rich domain, which may be a potential mechanism for regulating acyltransferase specificity and/or activity. S-acylation may tether cytoplasmic acylprotein thioesterase-1 to membranes, thus facilitating its interaction with and deacylation of membrane-associated S-acylated proteins. Our findings also suggest that certain ribosomal proteins may be targeted to lipid rafts via S-acylation, possibly to facilitate regulation of ribosomal protein activity and/or dynamic synthesis of lipid raft proteins in situ. In addition, bioinformatic analysis suggested that S-acylated proteins are highly enriched within core complexes of caveolae and tetraspanin-enriched microdomains, both cholesterol-rich membrane structures. The PalmPISC approach and the large-scale human S-acylated protein dataset are expected to provide powerful tools to facilitate our understanding of the functions and mechanisms of protein S-acylation.