O-Glycosylation is an increasingly recognized modification of intracellular proteins in all kingdoms of life, and its occurrence in protists has been investigated to understand its evolution and its roles in the virulence of unicellular pathogens. We focus here on two kinds of glycoregulation found in unicellular eukaryotes: one is a simple O-fucose modification of dozens if not hundreds of Ser/Thr-rich proteins, and the other a complex pentasaccharide devoted to a single protein associated with oxygen sensing and the assembly of polyubiquitin chains. These modifications are not required for life but contingently modulate biological processes in the social amoeba Dictyostelium and the human pathogen Toxoplasma gondii, and likely occur in diverse unicellular protists. O-Glycosylation that is co-localized in the cytoplasm allows for glycoregulation over the entire life of the protein, contrary to the secretory pathway where glycosylation usually occurs before its delivery to its site of function. Here, we interpret cellular roles of nucleocytoplasmic glycans in terms of current evidence for their effects on the conformation and dynamics of protist proteins, to serve as a guide for future studies to examine their broader significance.