Palmitoylation, a unique reversible post-translational lipid modification, adds the palmitate to proteins on specific cystein residues and regulates protein trafficking and functions. We identified the DHHC palmitoylating enzyme family in 2004 and have obtained important insights into various cellular functions since then. However, the significance of palmitoyl cycles remains incompletely understood because authentic depalmitoylating enzymes have been unidentified over 35 years. Here, to identify depalmitoylating enzymes, we focused on the uncharacterized serine hydrolase family including protease, lipase, esterase and thioesterase because depalmitoylating enzymes belong to thioesterase and classical depalmitoylating enzyme candidates, APT1 (Lypla1), APT2 and PPT1, belong to serine hydrolase family. We isolated 38 unannotated serine hydrolase genes and examined their enzymatic activities by metabolic labeling of HRas with tritium-palmitate. We found that six candidate proteins robustly reduced palmitoylation levels of HRas. Further analysis revealed their substrate specificities toward representative palmitoylated substrates, GAP-43, Fyn, Lck, SNAP-25, Paralemmin, NCAM140 and PSD-95. Intriguingly, when we examined the localization of these six candidates, they differently localized at the plasma membrane, ER or cytosol. Using fluorescence recovery after photobleaching analysis and photoactivation method, we propose that individual depalmitoylating enzyme could regulate different steps of HRas trafficking acting on specific palmitoylated cysteine sites at different subcellular locations.