N-glycans are involved in various physiological functions and their structures diverge among different phyla and kingdoms. Insect cells mainly produce high mannose-type and paucimannose-type glycans but very few mammalian like complex-type glycans. However, many insects possess genes for proteins homologous to the enzymes involved in complex-type N-glycan synthesis in mammalian cells, and their N-glycosylation pathway is incompletely understood compared with that of mammals. Here, we cloned a candidate gene for beta-1,2-N-acetylglucosaminyltransferase II (GnTII), which is a Golgi-localized enzyme involved in a key step in the conversion to complex-type N-glycans, from silkworm Bombyx mori, and the gene was found to be expressed ubiquitously in the larval and pupal stages. In addition, recombinant B. mori GnTII was expressed as a soluble form using a silkworm-B. mori nucleopolyhedrovirus bacmid expression system. The recombinant enzyme exhibited similar pH and temperature dependency and the same substrate specificity as human GnTII, but deglycosylation with peptide:N-glycanase F did not affect its enzymatic activity. Compared with the structure of human GnTII, the amino acid residues involved in catalytic activity and substrate recognition are almost fully conserved in B. mori GnTII, which is consistent with its enzymatic properties. These results raised the possibility of mammalian-like complex-type N-glycan synthesis using the GnTII ortholog in silkworm. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.