Endohexosaminidase D, a family 85 glycoside hydrolase from S. pneumoniae and the first endohexosaminidase to be discovered, is found to be capable of catalysing the glycosylation of a glycosyl amino acid bearing a GlcNAc residue using a variety of N-glycan oxazoline donors. Although enzyme-catalysed oxazoline hydrolysis is a significant competing reaction that is not countered by the addition of organic co-solvents or variation of reaction pH, a high yielding synthetic process can be achieved by the sequential addition of multiple equivalents of oxazoline donor, demonstrating the synthetic potential of this enzyme as a biocatalyst for the synthesis of defined glycoconjugates. Notably Endo-D does not appear to hydrolyse the resulting products under the conditions used. The synthetic activity displayed by Endo D implies that other, as yet untested, family GH85 enzymes may display similar synthetic potential. Furthermore since Endo D is capable of cleaving N-glycans attached to monoclonal antibodies (mAbs), and also of cleaving glycans that are core-fucosylated, the development of Endo D as a useful biocatalyst for the synthesis of important defined homogeneous complex glycoconjugates may have significant future potential, provided that the limitation of direct oxazoline hydrolysis can be surmounted.