In this study, we found that thermostable alpha-glucan phosphorylase from Aquifex aeolicus VF5 catalyzed enzymatic successive alpha-mannosylations of maltotriose using alpha-D-mannose 1-phosphate as a glycosyl donor to produce nonnatural heterooligosaccharides composed of alpha-(1 -> 4)-linked oligo-mannose residues at a nonreducing end of maltotriose.