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Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, which has been considered as one of the key targets for cancer therapy. However, currently approved therapeutic anti‐EGFR antibody may cause the hypersensitivity reaction induced by galactose‐α‐1,3‐galactose (α‐Gal) structure, which is inevitable in insect cell expression system. In this study, the Chinese hamster ovary cell line was used to produce a monoclonal antibody containing simplified glycosylation patterns (code: AB01). And cetuximab was used as a control. The two antibodies were highly similar in molecular weight, secondary structure, binding affinity and endocytosis behavior, whereas the glycotypes are extremely distinct. The flow cytometry assay suggested that AB01 induced cell cycle arrest in G1, thus inhibit cell proliferation. Moreover, both cetuximab and AB01 showed similar sensitivity for all tested cell lines in this research. In conclusion, AB01 could be a potential anti‐EGFR drug candidate for cancer therapy.