Protein palmitoylation, by modulating the dynamic interaction between protein and cellular membrane, is involved in a wide range of biological processes, including protein trafficking, sorting, sub-membrane partitioning, protein-protein interaction and cell signaling. To explore the role of protein palmitoylation in adipocytes, we have performed proteomic analysis of palmitoylated proteins in adipose tissue and 3T3-L1 adipocytes and identified more than 800 putative palmitoylated proteins. These include various transporters, enzymes required for lipid and glucose metabolism, regulators of protein trafficking and signaling molecules. Of note, key proteins involved in membrane translocation of the glucose-transporter Glut4 including IRAP, Munc18c, AS160 and Glut4, and signaling proteins in the JAK-STAT pathway including JAK1 and 2, STAT1, 3 and 5A and SHP2 in JAK-STAT, were palmitoylated in cultured adipocytes and primary adipose tissue. Further characterization showed that palmitoylation of Glut4 and IRAP was altered in obesity, and palmitoylation of JAK1 played a regulatory role in JAK1 intracellular localization. Overall, our studies provide evidence to suggest a novel and potentially regulatory role for protein palmitoylation in adipocyte function.