Human proteins destined for use as therapeutics, such as Monoclonal antibodies (mAbs), are expensive to produce using mammalian cell culture technoloogies. Transgenic platforms such as Bombyx mori silkworms could provide a more cost-effective method. The B. mori silk gland provides an ideal location for expression due to its lack of allergenic glycoforms and N-glycan core fucosylation, both of which are common in mammalian expression systems and are detrimental to proper function and in vivo tolerance . However, silkworms lack the complex glycosylation required for creation of mAb with increased thermostability and in vivo avidity to receptors. Recently functional recombinant mAbs and mammalian glycoenzymes, N-acetylglucosaminyltransferase (MGAT2) and β-1,4-galactosyltransferase (B4GALT1), have been expressed in the silk glands, and co-expressed in the hemolymph through the silkworm-baculovirus expression system, but have never been co-expressed in B. morisilk gland. We tested the hypothesis that anti-HER2 mAb (HER) co-expressed in the B. mori middle silk gland with MGAT2 and B4GALT1 would exhibit extended glycosylation leading to improved protein stability and greater efficacy of the mAb in both the ADCC and CDC pathways. To create transgenic silkworm lines, we co-injected mRNA expressing the piggyBac transposase and bacteria plasmids with piggyBac-flanked expression cassettes containing MGAT2/B4GALT1 glycoenzyme exons and anti-HER2 mAb, under the control of a silkworm sericin promoter. Expression cassette insertion of anti-HER2 mAb and MGAT2/B4GALT1 into the silkworm germlines was identified after 2 generations through use of co-expressed fluorescent eye markers. MGAT2/B4GALT1 functionality and novel mammalian glycosylation was tested by cleaving N-glycans from extracted total protein and isolated mAb and analyzing on a MALDI-TOF mass spectrometer. anti-HER2 mAb was extracted from silk gland and purified with Protein G columns and desalted. Avidity of silkworm-expressed anti-HER2 mAb to FcyRIIIa and C1q receptors was compared using ELISA and surface plasmon resonance. MGAT2/B4GALT1 expression in B. mori middle silk gland resulted in significant increase of mono- and biantennary terminal galactosylation on total proteins. Co-expression of MGAT2/B4GALT1 and anti-HER2 mAb produced only higher levels of biantennary N-acetylglucosamine (Gnt2) terminal ends on N-glycosylated mAb. As expected, Gnt2-terminal mAb had no improved binding to FcyRIIIa, while C1q binding was unexpectedly decreased. We hypothesize misfolding of mAb decreased C1q binding, and either steric hindrance of the mAb Asn-297 hydrophobic pocket or competition of other target substrates for B4GALT1 prevented significant galactosylation of anti-HER2 in B. mori MSG.