N-Glycosyltransferase (NGT) is an inverting glycosyltransferase for an unusual pathway of N-linked protein glycosylation and glycosylates polypeptides in the consensus sequon (N-(X≠P)-T/S) with hexose monosaccharides. Here， we expressed and characterized a novel N-glycosyltransferase from Aggregatibacter aphrophilus (named AaNGT). RP-HPLC and Mass Spectrometry were used to assay and quantify glycopeptide formation by AaNGT and determine its substrate specificities. AaNGT could utilize a variety of nucleotide-activated sugar donors， including UDP-Glc， UDP-Gal， UDP-Xyl， GDP-Glc， dGDP-Glc and UDP-GlcN， to glycosylate the tested peptides. To the best of our knowledge， AaNGT was the first identified natural glycosyltransferase able to transfer GlcN moiety onto asparagine residues. AaNGT also exhibited a different position-specific residue preference of substrate peptides from the NGT of Actinobacillus pleuropneumoniae (ApNGT). In vitro assays with diverse synthesized peptides revealed that AaNGT preferred different peptide substrates from ApNGT. The efficient glycosylation of natural short peptides by AaNGT showed its potential to modify important therapeutic mammalian N-glycoproteins.