Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core beta-1,2-xylose and alpha-1,3-fucose) and a Lewis a (Le(a)) epitope, Gal beta(1-3)[Fuc alpha(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core alpha-1,3-fucose and alpha-1,4-fucose residues in epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to Delta GMD plants) (Matsu and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core beta-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of (beta-1,2-xylosyltransferase (Delta XylT plant). By crossing AGMD and AXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (Delta GMD Delta XylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from Delta GMD Delta XylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in Delta GMD Delta XylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues. (c) 2014, The Society for Biotechnology, Japan. All rights reserved.