The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-beta-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced alpha-amylase with a molecular weight lower than that of native alpha-amylase without an effect on growth. To test whether the A. oryzae alpha-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged alpha-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.