The Notch signaling pathway is highly conserved and essential in animal development and tissue homeostasis. Regulation of Notch signaling is a crucial process for human health. Ligands initiate a signal cascade by binding to Notch receptors expressed on the neighboring cell. Notch receptors interact with ligands through their epidermal growth factor-like repeats (EGF repeats). Most EGF repeats are modified by O-glycosylation with residues, such as O-linked N-acetylglucosamine (O-GlcNAc), O-fucose, and O-glucose. A recent study revealed the distinct roles of these O-glycans in ligand binding, processing, and trafficking of Notch receptors. In particular, O-GlcNAc glycans are essential for Delta-like (DLL) ligand-mediated Notch signaling. In this study, we showed that O-GlcNAc promotes Notch1 trafficking to the cell surfaces under the condition that O-fucose and O-glucose are removed from consecutive EGF repeats of Notch1. Through in vitro experiments, we showed that O-GlcNAc mediates the stability of EGF domains in the same manner as O-fucose and O-glucose. Thus, O-GlcNAc on EGF domains possesses a shared function in the stability of EGF domains and Notch1 trafficking. (C) 2020 Elsevier Inc. All rights reserved.