A high-affinity receptor for interleukin (IL)-13 (interleukin-13R 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R2 (ECD2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD2 showed IL-13 inhibitory activities, mammalian cell-derived ECD2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin’s lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD2 was shown over ECD2/Fc fusion protein. The N-linked glycosylation of ECD2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD2 are receptor specific. These results indicate that glycosylated ECD2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.--Kioi, M., Seetharam, S., Puri, R. K. N-linked glycosylation of IL-13R2 is essential for optimal IL-13 inhibitory activity.