Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on N-linked oligosaccharides (NLO) of cell wall proteins in the hyphae of two developmental stages, vegetative mycelium and fruiting body. High mannose-type glycans were the most commonly found structures. In addition, we observed a novel glycan, predominantly present in fruiting body. This oligosaccharide structure was of the high mannose type with at least five mannoses and a bisecting alpha1-4 N-acetylglucosamine (GlcNAc) at the beta-mannose of the N-glycan core. The transferase responsible for this modification, CcGnt1 (C. cinerea GlcNAc transferase 1), was identified and expressed in insect cells. In vitro activity of CcGnt1 was demonstrated. This novel glycosyltransferase belongs to the glycosyltransferase family 8 (GT8) and is predicted to be a type II membrane protein. Expression of the CcGnt1 locus was up-regulated in fruiting body, but down-regulation of expression by means of RNAi decreased the level of bisected NLO; however had no apparent effect on fruiting body formation.