Glycosylation, a significant form of post-translational modification (PTM) in organisms, is aberrantly expressed in cancer due to altered glycosyltransferase activity. Studies have shown specific changes in glycan structures associated with epithelial-mesenchymal transition (EMT) of cancer cells. This study analyzed glycans in bronchoalveolar lavage fluid (BALF) from lung adenocarcinoma (LUAD) patients and found a significant reduction in glycans containing the bisecting-GlcNAc structure. Further investigation revealed that reduced expression of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3) downregulates epithelial markers, promoting EMT. Additionally, we observed a notable downregulation of both mRNA and protein expression of Forkhead box protein A2 (FOXA2) in early-stage LUAD, with FOXA2 loss emerging as an adverse prognostic indicator. Cellular models demonstrated that FOXA2 deficiency decreased MGAT3 expression during TGF-β1-driven EMT, leading to reduced levels of bisecting-GlcNAc N-glycans in LUAD cells. Our findings unveil a novel mechanism underlying the downregulation of MGAT3 and bisecting GlcNAc N-glycan expression during EMT, a process crucial for tumor metastasis.