Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of alpha and beta subunits. Here we present the first characterization of a plant glucosidase II alpha subunit at the molecular level. Expression of the Arabidopsis alpha subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe alpha- or alphabeta-deficient mutants, but with a lower efficiency in the last case. Inactivation of the alpha subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development.