Myeloperoxidase (MPO), which predominantly resides in the primary granules of resting neutrophils but reportedly also in other neutrophil compartments at lower levels, is an important glycoprotein in neutrophil-mediated innate immunity. Most structural features of human neutrophil MPO and its capacity to produce anti-microbial hypochlorous acid are well-documented, but the exact site- and granule-specific glycosylation of MPO remains undocumented, despite the recognised importance of N-glycosylation for its structure-function. Herein, we performed a deep structural characterisation of the N-glycosylation of human MPO from healthy donors using LC-MS/MS-based glycomics, intact glycopeptide and glycoprotein profiling. Quantitative glycomics and glycopeptide analyses of MPO from neutrophil lysates revealed that all five N-glycosylation sites of the heavy chain MPO monomer are conjugated with heterogeneous N-glycans albeit with strong site-specific differences. Paucimannosidic N-glycans were predominantly found to be linked to Asn323 (47%) and Asn483 (56%), oligomannosidic N-glycans were mainly carried by Asn355 (97%) and Asn391 (64%) while Asn729 was largely unoccupied (44%) or modified with chitobiose core N-glycans (33%). Importantly, the glycoprofiles of the intact MPO monomer and dimer obtained using high-resolution top-down mass spectrometry recapitulated the heterogeneity observed at the bottom-up level as supported by matching experimental and theoretical intact glycoprotein masses. Further, glycoprofiling of MPO from isolated neutrophil granules intriguingly demonstrated that the N-glycosylation of Asn355, Asn391 and Asn729 varied dramatically whereas Asn323 and Asn483 were similarly glycosylated on MPO across the studied neutrophil compartments. Particularly the abundant primary granule-resident MPO displayed unique glycosylation signatures comprising a higher proportion of oligomannosidic N-glycans on the three variable glycosylation sites. In conclusion, this study represents the most detailed structural characterisation of MPO N-glycosylation to date. The complex site- and granule-specific N-glycosylation of MPO, shown here for the first time, adds fundamental knowledge that aid our understanding of the fascinating glycobiology underpinning neutrophil-mediated immunity.