Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has transglycosylation activity, and high-mannose-type oligosaccharides are transferred to suitable glycosides as acceptor substrates. The acceptor specificity of Endo-A-catalyzed transglycosylation toward various disaccharides was investigated. To identify an effective acceptor for the transglycosylation by Endo-A, the reaction was carried out using various disaccharides. Endo-A transferred high-mannose-type oligosaccharides more efficiently to β-linked disaccharides (cellobiose, gentiobiose, sophorose, and laminaribiose) than to α-linked disaccharides (isomaltose, maltose, nigerose, kojibiose, and trehalose) as acceptor substrates. The transglycosylation products, (Man) 6 GlcNAc-Glc-β-Glc, were more rapidly hydrolyzed than (Man) 6 GlcNAc-Glc-α-Glc. These results indicate that Endo-A recognizes the anomeric configuration of the acceptor substrates, and β-linked glycosides are suitable for the synthesis of transglycosylation products.