The transport of proteins between the different cellular compartments and the cell surface is governed by the secretory pathway. Alternatively, unconventional secretion pathways have been described in mammalian cells, especially through multivesicular bodies and exosomes. These highly sophisticated biological processes rely on a wide variety of signaling and regulatory proteins that act sequentially and in a well-orchestrated manner to ensure the proper delivery of cargoes to their final destination. By modifying numerous proteins involved in the regulation of vesicular trafficking, post-translational modifications (PTMs) participate in the tight regulation of cargo transport in response to extracellular stimuli such as nutrient availability and stress. Among the PTMs, O-GlcNAcylation is the reversible addition of a single N-acetylglucosamine monosaccharide (GlcNAc) on serine or threonine residues of cytosolic, nuclear, and mitochondrial proteins. O-GlcNAc cycling is mediated by a single couple of enzymes: the O-GlcNAc transferase (OGT) which catalyzes the addition of O-GlcNAc onto proteins, and the O-GlcNAcase (OGA) which hydrolyses it. Here, we review the current knowledge on the emerging role of O-GlcNAc modification in the regulation of protein trafficking in mammalian cells, in classical and unconventional secretory pathways.