Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein with three reported N-glycosylation sites, which integrates many biological processes such as antiangiogenic activity, immune complex clearance and pathogen clearance. Importantly, the protein is known to have five genetic variants with minor allele frequencies of more than 10%, meaning these exist with substantial frequency in the human population. Among them, Pro204Ser can induce a new N-glycosylation site at Asn202. Considerable research has been performed into the biological activity of HRG, while research on its glycosylation is rare. To close this knowledge gap, we used C18-based LC-MS/MS to investigate the glycosylation characteristics of HRG from human plasma, recombinant Chinese hamster ovary (CHO) cell lines and recombinant human embryonic kidney (HEK293) cell lines with targeted mutations. Within human plasma endogenous HRG, every N-glycosylation site proved dominant with a sialylated diantennary complex-type glycan. For the recombinant HRGs, on the other hand, glycans showed different antennarities, sialylation and core-fucosylation, as well as the appearance of oligomannose glycans, LacdiNAc and antennary fucosylation. Furthermore, we discovered a previously unreported O-glycosylation site on residues Thr273/Thr274, which showed an approximate 90% glycan occupancy in all HRG types. To investigate the relevance of HRG glycosylation characteristics and its biological function, we set up an assay to study the plasmin cleavage of HRG under various conditions. In doing so, we showed that the sialylation of the new O-glycan, as well as the genotype-dependent N-glycosylation, significantly influenced the plasmin cleavage of HRG.