High-mannose glycans, containing 69 mannose residues, are favorable for in vitro mannose phosphorylation to produce mannose-6-phosphate (M6P) residues, which are essential for uptake into target cells during enzyme replacement therapy of Pompe disease. Golgi a-mannosidase-I mediates mannose trimming in the N-glycosylation pathway. In this study, mutant rice with a T-DNA insertion in the Os04g0606400 (?a-manI) gene was used to produce recombinant human acid a-glucosidase (rhGAA). Expressed and secreted rhGAA from ?a-manI (?a-manI-GAA) in suspension culture media was purified, and analysed by MALDI-TOF. It showed the presence of high-mannose type N-glycans with Man8 (63.8%), Man7 (22.5%), and Man6 (5.4%).