Chemo-enzymatic addition of a high-mannose type oligosaccharide to eel calcitonin (CT), a calcium-regulating hormone, was examined. The endo-beta-N-acetylglucosaminidase ase from Arthrobacter protophormiae (Endo-A) transglycosylated the Man(6)-GlcNAc moiety from an ovalbumin-derived high-mannose type glycosyl asparagine, Asn(Man(6)-GlcNAc(2))-OH, to a calcitonin derivative, [Asn(GlcNAc)(3)]-CT, in which the N-acetyl-D-glucosamine (GlcNAc) is attached to the third L-asparagine (Asn) residue of the peptide, and a calcitonin derivative containing a high-mannose type oligosaccharide, [Asn(Man(6)-GlcNAc(2))(3)]-CT, was synthesized. The optimal reaction conditions for the synthesis of [Asn(Man(6)-GlcNAc(2))(3)]-CT from Asn(Man(6)-GlcNAc(2)-OH) and [Asn(GlcNAc)(3)]-CT catalyzed by Endo-A were examined. The transglycosylation yield relative to the concentration of the [Asn(GlcNAc)(3)]-CT added was 32.7%, and 4.42 mg of [Asn(Man(6)-GlcNAc(2))(3)]-CT was prepared.