Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid-sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site-specific characterization of the N- and O-linked glycosylation of serum-derived hSHBG. MS-driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom-up and top-down manner to determine glycosylation sites, site-specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum-derived hSHBG is N-glycosylated at Asn351 and Asn367 with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi- and triantennary N-Glycoforms with lactosamine-type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N-Glycoforms of Asn367 were slightly more branched and core fucosylated than Asn351 N-glycoforms due probably to a more surface-exposed glycosylation site. The N-terminal Thr7 was fully occupied by the two O-linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn351 glycan occupancies and N-glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.