Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal storage disorder (LSD) caused by recessive mutations in the α-L-iduronidase (IDUA) gene. Enzyme replacement therapy (ERT) utilizing terminal mannose 6-phosphate (M6P)-carrying N-glycans attached to therapeutic enzymes produced by mammalian cell lines has been clinically applied to several LSDs. Recent studies suggested an unidentified delivery pathway mediated by sialic acid-containing N-glycans. However, more economical platform development is required to produce large quantities of recombinant enzymes. Transgenic silkworms have been established as low-cost systems for expressing recombinant glycoproteins. Microbial endo-β-N-acetylglucosaminidases (ENGases) enable the transglycosylation of N-glycans to other types. In the present study, we established IDUA transgenic silkworms and purified recombinant human IDUA from cocoons. As M6P- and sialic acid-containing N-glycans were not attached to purified hIDUA, we performed ENGase-mediated transglycosylation to obtain hIDUAs with M6P- and sialic acid-containing N-glycans (neoglyco-hIDUAs). Furthermore, we performed intravenous neoglyco-hIDUA replacement therapy in a Japanese macaque MPS I non-human primate model carrying a homozygous Idua missense mutation and succeeded in improving the clinical symptoms and reducing the urinary glycosaminoglycan (GAG) levels. These glycotechnologies using transgenic silkworms and ENGases are expected to serve as platforms for developing therapeutic glycoproteins.