Using plants as production factories for therapeutic proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In an attempt towards such humanization, we disrupted the genes for α1,3-fucosyltransferase and β1,2-xylosyltransferase in Physcomitrella patens by homologous recombination. The single Δfuc-t and Δxyl-t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild-type moss in morphology, growth, development, and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF121, into the culture medium. N-Glycan analysis, however, revealed the absence of 1,3-fucosyl and/or 1,2-xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant-made glycosylated biopharmaceuticals with nonallergenic N-glycans.