Human NOTCH1 receptor contains 36 epidermal growth factor (EGF)‐like repeating domains, in which O‐glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O‐glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor–ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O‐glycosylation patterns on the pivotal EGF‐like repeats 10, 11, and 12, we performed chemical synthesis and NMR studies of site‐specifically O‐glycosylated EGF11 and EGF10. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O‐glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type “sugar bridges” crosslinking Thr‐O‐GlcNAc in the consensus sequence C5‐X‐X‐G‐X‐(T/S)‐G‐X‐X‐C6 and an amino acid in the hinge region between the domains, 445Thr‐O‐GlcNAc—IIe451 in domain 11 and 405Thr‐O‐GlcNAc—Gln411 in domain 10, respectively.