Summary: A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N‐glycosylation (i.e. the presence of β1,2‐xylosylation and core α1,3‐fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down‐regulation of the endogenous β1,2‐xylosyltransferase (XylT) and α1,3‐fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco‐related plant species widely used for recombinant protein expression. Three glyco‐engineered lines with significantly reduced xylosylated and/or core α1,3‐fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild‐type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3‐fucose residues in their N‐glycans were produced. Notably, 2G12 produced in XylT/FucT‐RNAi plants was found to contain an almost homogeneous N‐glycan species without detectable xylose and α1,3‐fucose residues. Plant‐derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)‐derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N‐glycan structure.