We have previously documented that human neutrophils are actively expressing immune-related glycoproteins carrying paucimannosidic glycans (Man1-3GlcNAc2Fuc0-1), a functional yet under-represented class of N-glycosylation in the human glycobiological literature. Whilst inference from the paucimannose-rich invertebrates and plants points to a β-N-acetyl-hexosaminidase (Hex)-driven production of paucimannosidic proteins from immature β1,2-linked N-acetylglucosamine-terminating glycoprotein intermediates in the mammalian glycosylation machinery, this hypothesis remains experimentally unsupported for human neutrophils. We therefore sought to obtain evidence for the involvement of the Hex isoenzymes, which exist in homodimeric (αα and ββ) and heterodimeric (αβ) variants arising from two coding genes, the HEXA (α) and HEXB (β), in the biosynthesis of paucimannosidic proteins in human neutrophils. Our approach was to perform quantitative PGC-LC-MS/MS-based N-glycomics of several CRISPR-Cas9-edited Hex-deficient undifferentiated neutrophil-like HL-60 mutant cell lines (one HEXA-/- and three HEXB-/-) relative to unedited HL-60 control cells. Accurate disruption of the two target genes and absence of off-target genetic mutations were validated using next-generation sequencing and a conventional 4-methylumbelliferyl-(6-sulfo-)N-acetyl-β-glucosaminide-based Hex activity assay. Importantly, the N-glycome of all four Hex-deficient mutants displayed a significant reduction of paucimannosidic N-glycans (2.2-3.6%) relative to unedited HL-60 (13.8%, p<0.05). In particular, Man2-3GlcNAc2Fuc0-1 displayed a significant reduction in the Hex-deficient mutants relative to unedited HL-60. Interestingly, an N-glycomics follow-up analysis of mature blood neutrophils from a patient diagnosed with early onset Sandhoff disease (HEXB-/-) recapitulated a reduced expression level of paucimannosidic N-glycans (20.7%) relative to levels found in mature blood neutrophils from a healthy age-matched donor (40.5%). This study provides evidence to support that several Hex isoenzyme variants encoded by both HEXA and HEXB are directly responsible for the biosynthesis of paucimannosidic proteins in human neutrophils. These well-characterised Hex-deficient mutant cell lines displaying low protein paucimannosylation may also be useful to further our understanding on the functional roles of paucimannosidic proteins in neutrophil glycoimmunology.