Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase frequently modified by glycosylation post-translationally, and in cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed a glycosylation-deficient mutant, EGFR N361A, with or without the oncogenic mutation L858R, and immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR and also reduced sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 revealed that N361A mutations increased co-localization. Located near the binding interface for the EGFR inhibitor necitumumab, N361A desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. Disruption of glycosylation at N361, near the ligand binding and dimerization regions, created a dominant negative form of EGFR that non-productively co-localized with HER2, resulting in a blockage in proliferation. These findings underline the critical relevance of post-translational glycosylation modifications on EGFR function.